Monitoring and inhibiting human immunodeficiency virus infection by modulating hmgb1 dependent triggering of hiv-1 replication and persistence

ABSTRACT

Compositions and methods for modulating human immunodeficiency virus (HIV) infection involving substances that inhibit the ability of high mobility box 1 (HMGB1) protein to interact with natural killer (NK) cells. Therapeutic compositions comprising antibodies and drugs, such as glycyrrhizin, which bind to HMGB1. Methods of detecting or monitoring HIV infection involving detection or quantitation of HMGB1 or antibodies specific for HMGB1 in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application61/096,135, filed Sep. 11, 2008 which is incorporated by reference. Thecorresponding PCT application which claims priority to U.S. U.S.Provisional Application 61/096,135 is also incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Diagnostic and prognostic methods involving measuring HMGB1 levelsand/or antibodies specifically raised against HMGB1. Antibody- anddrug-based methods for treating or reducing the severity of humanimmunodeficiency infection by modulating the activity of HMGB1.

2. Description of the Related Art

Early stages of HIV-1 infection are associated with local recruitmentand activation of important effectors of innate immunity, NK cells andDCs. In the first hours and days of mucosal infection, HIV-1 crosses theepithelial barrier and infects CCR5-expressing DCs, macrophages and Tcells in the mucosal tissues to initiate infection^(1, 2). DCs expressCD4, CCR5, DC-SIGN³ and other C-type lectin receptors (CLRs) thatfacilitate capture and dissemination of HIV-1^(4, 5). Immature DCs(iDCs) capture HIV-1 through CLRs⁶ and captured virus can beinternalized and rapidly transmitted to nearby CD4 T cells, in the formof an infectious synapse^(7, 8). DC-T cell conjugates facilitateproductive infection in CD4 T cells⁹, and dissemination of the infectionto the draining lymph nodes and subsequent other lymphoid tissuecompartments is ensured by virus-carrying DCs together with infectedmacrophages and CD4 T cells¹⁰.

Migration of iDC to T cell area of secondary lymphoid tissues aftervirus uptake is associated to a maturation process that allows theresulting mature DC (mDC) to prime an antigen-specific response¹¹.Recently, the fate of DCs has been found to be extremely dependent onautologous NK cells¹². NK-iDC interaction results in activation of NKcells that, in turn, induces DC maturation or killing, depending ontheir respective density^(13, 14, 15). DC undergoing maturation secreteseveral cytokines, such as IL-12 and IL-18, that act as potent inducersof NK cell activation and cytotoxicity^(16, 17, 18, 19, 20). In turn,once activated, NK cells produce IFN-γ and TNF-α, capable of inducing DCmaturation. This phenomenon is dependent on the engagement of NKp30 byligands expressed on iDC^(17, 21), and the down-regulation on iDC ofHLA-E, the ligand for CD94/NKG2A inhibitory receptor²². Anothermechanism was proposed suggesting that NK cells, activated by IL-18released by iDC at the synaptic cleft, secrete HMGB1, which induces DCmaturation and protects DCs from lysis²⁰. HMGB1 is a nuclear proteinthat is present in almost all eukaryotic cells, and it functions tostabilize nucleosome formation, and acts as a transcription-factor-likeprotein that regulates the expression of several genes^(23, 24). HMGB1is released from necrotic cells, but it can also be secreted byactivated macrophages²⁵ and activated NK cells²⁰ in response toinflammatory stimuli, and it is one of the main prototypes of thedamage-associated molecular pattern molecules (DAMPs)²⁶. It was recentlydiscovered to be a crucial cytokine in the immune system, facilitatingthe trafficking of inflammatory leukocytes, and being critical for DCsto mature, reach the lymph nodes and sustain the proliferation ofantigen-specific T cells, and to promote their polarization towards aT-helper 1 phenotype^(27, 28).

The mechanisms involved in NK-DC interaction during viral infections arepoorly understood. It was recently reported in murine CMV (MCMV)infection that MCMV-infected DCs were capable of activating syngeneic NKcells in vitro and also capable of enhancing NK-cell dependent clearancein vivo²⁹, demonstrating the crucial role of NK-DC cross-talk incontrolling viral replication. In HIV infection, NK-DC interaction wasfound defective in HIV-1-infected viremic, but not aviremic patients,characterized by abnormalities in the process of reciprocal NK-DCactivation and maturation, as well as a defect in NK-cell elimination ofiDCs³⁰. The role of NK-DC cross-talk on maturation, function, andsusceptibility to viral replication of HIV-1-infected iDCs wasevaluated. It was discovered that maturation of HIV-1-infected DCs couldbe triggered by activated NK cells, but it was associated with a strongimpairment of mature infected DCs to induce Th1 polarization followingtheir crosstalk with NK cells. In addition, the cross-talk between NKcells and HIV-1-infected iDCs resulted in a dramatic increase in viralreplication and proviral DNA expression in DCs. This process was mainlytriggered by HMGB1, released both by NK cells and DCs, as a consequenceof NK-DC cross-talk.

HIV-1 has evolved ways to exploit DCs, thereby facilitating viraldissemination and allowing evasion of antiviral immunity. The fate ofDCs is dependent on NK cells. Below, the inventors detail the impact ofNK-DC crosstalk on the fate of HIV-1-infected DCs. Activated NK cellsefficiently triggered maturation of infected DCs, but this wasassociated with a strong impairment of mature DCs to induce Th1polarization. Moreover, the crosstalk between NK cells and infected DCsresulted in a dramatic increase in viral replication and HIV-DNA in DCs.HMGB1 was crucial in this process, and inhibition of HMGB1 activity byglycyrrhizin or specific antibodies abrogated HIV-1 replication in DCs.The inventors describe how their new insights about how HIV ‘hijacks’DCs to promote efficiently viral dissemination can provide new ways toinhibit HIV infection, new ways to diagnose and monitor HIV infection,new ways to monitor HIV infection, the viral load and the efficiency oftreatment directed against HIV infection and new ways to carry out theprognosis of the state of progression of AIDS or towards AIDS.

DESCRIPTION OF THE INVENTION

Aspects of the invention include the following therapeutic, prognosticand diagnostic applications.

Blocking HMGB1 in patients can help suppress HIV replication, decreaseHIV reservoirs in DCs and slow down disease progression. Thus, oneaspect of the invention involves a method for modulating humanimmunodeficiency virus (HIV) infection comprising contacting a subjectinfected by HIV with an agent that binds to HMGB1, in particular anantibody that binds to High mobility group box 1 protein (HMGB1) or anHMGB1-binding antibody fragment, glycyrrhizin or the isolated RAGE or afragment of RAGE able to bind HMGB1. The invention also concerns anagent that binds to HMGB1, in particular an antibody that binds to Highmobility group box 1 protein (HMGB1) or an HMGB1-binding antibodyfragment, glycyrrhizin or the isolated RAGE or a fragment of RAGE ableto bind HMGB1, for use as a drug to treat HIV infection in a subjectinfected by HIV. A particular agent that may be used in therapy is anantibody specifically blocking HMGB1 or a fragment of such antibody, inparticular an antibody fragment which retains said ability tospecifically block HMGB1. Examples of fragments are a single-chainantibody, or a Fab, Fv and Fab₂ fragment. In a particular embodiment,said antibody is a monoclonal antibody, or said fragment is a part of amonoclonal antibody. In another particular embodiment, said antibody orfragment is preferably human or humanized. By “specifically blocking”,it is meant that the antibody or fragment thereof has the ability tobind the HMGB1 protein and prevents or decreases its activity, inparticular to prevent its binding on at least one of its receptors, inparticular the RAGE receptor. In a particular embodiment, the occurrenceof the blocking behavior of the antibodies of the invention or theirfragments may be tested either by assaying the binding of HMGB1 on atleast one of its receptors, and/or by assaying the activity of HMGB1 ondendritic cell (DC) maturation (whether HIV-infected or not), on HIVreplication in DC and/or on HIV DNA expression in DC. An antibody orfragment thereof is considered to specifically block the HMGB1 protein,when the decrease of the binding of HMGB1 on one of its receptors (inparticular RAGE) or the decrease of the activity of HMGB1 as definedabove is more than 50%, more than 60%, more than 70%, more than 80% ormore than 90%.

In the context of the invention, the term “specifically” or “specific”means that the antibodies or their fragments are able to recognize andto bind the HMGB1 protein, preferably to other cellular proteins and inparticular do not significantly recognize and bind other cellularproteins involved in the immune system, in particular in the context ofthe NK-DC cross-talk or do not significantly recognize and bind othercellular proteins. In the present application, unless otherwise stated,description relating to antibodies applies to their fragments asdisclosed above.

While not being bound to a particular mechanism of action, this methodmay operate by reducing viral replication and replenishment of viralreservoirs in dendritic cells. Thus, the invention also relates to anagent that binds to HMGB1 as mentioned above for use as a drug todecrease the HIV-reservoir cells, in a subject infected by HIV. TheHIV-reservoir cells may be any cell that is sensitive to the HIV and/orcan be infected by the HIV. In a particular embodiment, the HIVreservoir cells harbor the proviral DNA. The HIV reservoir cellsoriginate from biological tissues such as blood, solid tissues ormucosa, and in particular from brain, liver, spleen, tonsils, nodes orgut-associated lymphoid tissue (GALT). In a particular embodiment, thesecells are peripheral blood cells, lymphoid lineage cells such as T cellsespecially T CD4 cells, or are monocyte-derived cells such asmacrophages or dendritic cells.

Human immunodeficiency virus includes both HIV-1 and HIV-2 strains aswell as other variants of this virus, including HIV strains adapted tosimians and other mammals.

The invention also applies to treatment, diagnosis and monitoring ofinfections caused by other retroviruses, including HIV-2 and simianimmunodeficiency virus (SIV). Subjects or patients infected byretroviruses like HIV include humans, monkeys and other simians, andother mammals used models of HIV infection. Specific HIV-1 strainsinclude the R5 HIV-1 strain and the X4 HIV-1 strain.

HMGB1 is a well-known protein appearing in the nucleus and is also knownto be a cytokine. Physical and functional characteristics of HMGB1 aredisclosed by and incorporated by reference to Lotze, et al., NatureReviews, Immunology 5:351 (2005).

Antibodies which bind to HMGB1 are known and can be produced by methodswell-known in the art. An example of commercially available anti-HMGB1antibodies are Rabbit primary polyclonal antibodies to human HMGB1(Abcam ref. 18256) which are directed against a KLH-conjugated syntheticpeptide derived from residues 150 to C-terminus of human HMGB1. Thesemethods include those which produce polyclonal antibodies to HMGB1 andmonoclonal antibodies to HMGB1 or to specific fragments of HMGB1.Antibodies used in therapeutic applications have the characteristic tobe blocking, e.g., especially they interfere with HMGB1-induced HIVreplication in infected dendritic cells. These antibodies are preferablyderived from the same species as the subject to which they areadministered and recognize or are induced to the HMGB1 of the samespecies to which they will be administered. These antibodies may havedifferent isotypes, such as IgA, IgG or IgM isotypes. Antibody fragmentswhich bind HMGB1 may also be employed, including Fab, Fab₂, and singlechain antibodies or their fragments.

Humanized anti-HMGB1 monoclonal antibodies may also be employedtherapeutically in human. These may be produced by methods well-known inthe art. Injection of these antibodies to HIV infected patients withhigh viral load and elevated levels of HMGB1 can be used to reduce virusreplication and limit the number of reservoir cells. Such humanizedantibodies, can be used as salvage or alternative therapy, or combinedwith antiretrovirals.

Antibodies or their fragments as defined herein that bind to HMGB1 maybe administered to a subject to bind to HMGB1 and modulate HIVreplication or infection in the subject. Modes of administrationinclude, but are not limited to, intravenous (i.v.), intradermal,subcutaneous (s.c.), intracerebral, transmucosal, transdermal, byinhalation (e.g., intratracheal, intrapulmonary, or intrabroncial),intransal, oral, subuccal, transdermal, and rectal administration.

Targeting HMGB1 production or release, or preventing its interactionwith its receptor(s), in particular RAGE on DCs, may be employed totreat chronic viral infections, considering its impact on theinflammatory response and maturation and survival of infected DCs. Thus,agents, such as antibodies or antibody fragments, which bind to HMGB1receptor, (e.g. RAGE) and inhibit its interaction with HMGB1 or solubleHMGB1 receptor proteins (e.g. soluble RAGE proteins or fragment able tobind HMGB1) that inhibit functional interaction of HMGB1 receptor (e.g.RAGE) on DCs and HMGB1 may be employed. Portions of HMGB1 that bind toRAGE on DC's and inhibit the functional interaction of HMGB1 with DCsare also contemplated.

The inventors have shown that glycyrrhizin is able to inhibitHMGB1-dependent HIV replication in DCs. Glycyrrhizin therapy has fewside effects and it has been recently used successfully in vivo toprevent hepatocellular carcinogenesis in patients with IFN-resistantactive chronic hepatitis C. This therapy may be used in chronicallyHIV-infected patients with detectable viral load and increased levels ofHMGB1, either as salvage therapy because of multi-drug resistance virus,as an alternative therapy (less toxic and acting on the inflammatorymicroenvironment rather than on the virus itself) to the use ofHIV-specific anti-retrovirals, or a combined therapy withanti-retrovirals in case of incomplete success of these drugs.

Therefore, yet a further aspect of the invention is a method formodulating human immunodeficiency virus (HIV) infection, including HIV-1infection, comprising contacting a cell or a subject infected by HIVwith an amount of an agent that binds to High mobility group box 1protein (HMGB1), in particular which inhibits natural killer (NK) celldependent triggering of HIV replication in a dendritic cell (DC). Theinvention also concerns an agent that binds to High mobility group box 1protein (HMGB1), in particular which inhibits natural killer (NK) celldependent triggering of HIV replication in a dendritic cell (DC), foruse in the modulation of human immunodeficiency virus (HIV) infection(including HIV-1 infection) in a cell or a patient infected by HIV.

Glycyrrhizin is one such agent and modes and concentrations ofglycyrrhizin useful for providing binding to HMGB1 are disclosed andincorporated by reference to Mollica, et al., Chem. Biol. 14:431 (2007).Other agents or compounds, besides glycyrrhizin, that bind to HMGB1 mayalso be employed. Soluble ligands or segments of natural ligands towhich HMGB1 binds may be employed. Such ligands may be obtained fromleukocytes or antigen-presenting cells to which HMGB1 binds.

Human patients infected with, or at risk of, HIV infection may betreated with the antibodies, antibody fragments, and other HMGB1-bindingagents disclosed herein in order to maintain the immune system of thepatients including with an antibody specifically blocking HMGB1 or afragment which retains said ability to specifically block HMGB1. Sinceglycyrrhizin is nontoxic while many antiretroviral drugs causesubstantial toxicity, the treatments of the invention can reducedetrimental side-effects of conventional anti-HIV therapy.Antibody-based products recognizing HMGB1 also lack the toxicity of manyanti-HIV drugs and can also be employed to reduce the side-effects ofHIV treatment. Similarly, advanced treatment of human patients who areresistant, or have developed multiple resistances to conventionalretroviral drug treatments can be treated with the methods of theinvention, including antibody products and other agents that bind toHMGB1. Combined therapy with glycyrrhizin (or humanized blockinganti-HMGB1 antibodies) and anti-retroviral drugs at lower doses thatwhen used alone is also contemplated.

These HMGB1 binding agents or compounds may be used alone or incombination with at least one further active compound against HIVinfection, or be administered in combination with other agents, such asdrugs and pharmaceutical agents, used to treat HIV infection. Examplesof such drugs and pharmaceutical agents include two nucleoside analoguereverse transcriptase inhibitors (NARTIs or NRTIs), protease inhibitors,and non-nucleoside reverse transcriptase inhibitors (NNRTIs), includingAZT and Indinavir.

The invention also includes sterile compositions, suitable foradministration to human subjects comprising an isolated antibody orantibody fragment that binds to High mobility group box I (HMGB1)protein, other HMGB1 binding agents, and/or glycyrrhizin; and apharmaceutically acceptable carrier, excipient, or diluent. Thesecompositions may contain other drugs or pharmaceutical agent other thansaid antibody, antibody fragment and/or glycyrrhizin, used to treathuman immunodeficiency virus infection, such as those mentioned above.Generally, antibodies used as therapeutic tool for HIV-infected humanpatients should be human or humanized antibodies which block theactivity of HMGB1.

Other aspects of the invention include glycyrrhizin for use as a drug totreat HIV infection in human, use of glycyrrhizin for the manufacture ofa medicament for therapeutic application in HIV infections, and use ofan isolated humanized blocking HMGB1-specific antibody or antibodyfragment for the manufacture of a medicament for the therapeuticapplication in HIV infections.

The invention also concerns an in vitro method for quantitating totalantibodies specific for HMGB1 contained in a biological sample obtainedfrom a subject, comprising (a) treating the sample by an acid treatmentto dissociate the immune complexes involving HMGB1 found in the sample,preferably with glycine 1.5M at a low pH; (b) contacting said treatedbiological sample with native HMGB1 protein or derivatives thereof; and(c) quantitating the total antibodies specific for HMGB1.

In a preferred embodiment, the acid treatment consists to put in contactthe sample with an acidic dissociation solution, having a low pH,preferably between pH 1 and 3, chosen to separate the HMGB1 protein fromantibodies to which it is immunologically bound in the sample, withoutaltering binding ability of this antibody. In a particular embodiment,the acidic dissociation solution is glycine (e.g. 1.5M) at a low pH,preferably between pH 1 and 3 (e.g. 1.85). The acid treatment is thenstopped with a neutralization buffer (such as Tris, for example 1.5MTris, pH9). In another preferred embodiment, in combination with theprevious one or not, the incubation with the acidic dissociationsolution is carried out at a temperature between 20 and 37° C.,preferably at 25° C., and/or the neutralization step takes place in ice.

In the present application, the term “quantitating” encompasses the term“quantifying” and any suitable informative determination of the HMGB1protein or specific antibodies.

The invention also relates to an in vitro method for monitoring the HIVinfection, in a biological sample obtained from a subject who is knownto be infected with HIV, comprising quantitating the antibodies specificfor High mobility group box I (HMGB1) contained in a biological sampleobtained from this patient, wherein the antibodies targeted forquantitation are either the total antibodies specific for HMGB1 or theircirculating fraction (circulating antibodies) or their immunologicalcomplexed fraction.

The methods for monitoring the HIV infection, the viral load or theefficacy of a treatment and the prognostic method disclosed herein, maybe implemented based on either the quantitation of the circulating(residual) antibodies specific for HMGB1, or on the quantitation of thetotal antibodies specific for HMGB1 or on the quantitation of thefraction of immunological HMGB1/specific antibodies complex.

In a particular embodiment, all these methods are based on either thequantitation of circulating specific antibodies or total specificantibodies.

The quantitation of the total antibodies specific for HMGB1 may bepreferred when the level of circulating antibodies specific for HMGB1 islow.

When the quantitation is based on the total antibodies specific forHMGB1, the methods of the invention also comprise a step suitable fordissociation of immunological complexes formed with HMGB1-specificantibodies, and for example the methods of the invention use or includethe quantitation method based on the acidic treatment as disclosed aboveand illustrated in the examples.

In a particular embodiment, said quantitation of the antibodies specificfor HMGB1 is carried out by contacting a biological sample (obtainedfrom a subject) with the High mobility group box I (HMGB1) protein orderivatives thereof. The contact of the sample with said antibody aswell as the quantification of the formed complex are carried out invitro.

The invention also concerns a method for monitoring the HIV viral loadin a biological sample obtained from subject, who is known to beinfected with HIV, comprising carrying out the method of quantitation ofthe total antibodies specific for HMGB1 or the method of monitoringbased on the antibodies specific for HMGB1 mentioned herewith, whereinthe more the antibodies specific for HMGB1, the less the viral load.

By “viral load”, it is meant either the HIV RNA (which is derived fromviral particles and present in plasma) or the HIV DNA (which isintegrated in the cell genome and present in cells). In a particularembodiment, the methods of the invention based on the quantitation ofantibodies specific for HMGB1 are suitable to monitor the HIV RNA viralload.

It is understood that for the quantitation method and the methods ofmonitoring the HIV infection, of the viral load or of the efficacy of atreatment and the prognostic method of the invention, it is possible touse the sequence of the full length HMGB1 protein (mammalian origin,preferably human origin) or any peptide (10 to 30 amino acid residues)or polypeptide (30 to 215 amino acid residues, preferably 30 to 50, orto 100, or 30 to 150 residues) derived from HMGB1 (HMGB1 proteinderivatives) as long as these derivatives bind to antibodies specificfor HMGB1 and/or enable to quantitate the anti-HGB1 antibodies. Suchderivatives are selected in the group consisting of a recombinant HMGB1(e.g. HMG biotech, HM-115), an immunologically reactive part of HMGB1,an immunologically reactive part of HMGB1 whose sequence is common toHMGB1 proteins of various origins. Such an example is the recombinantBOXB from HMGB1 corresponding to the sequence common to human and mouseof HMGB1 (HMGbiotech HM-051).

These methods are carried out on a biological sample obtained from asubject infected with HIV, such as blood, plasma, serum, saliva, or anybody fluid or tissue.

By “monitoring the HIV infection”, it means the comparison of theprogression of the HIV infection, i.e. the decrease, the increase or thestability, as compared to a previous assay. The progression of the HIVinfection reflects the HIV replication and/or the integration of the HIVgenome into the genome of target cells.

The invention also relates to an in vitro method for monitoring theefficacy of a treatment directed against HIV infection, in a subjectinfected with HIV comprising carrying out the method of quantitation ofthe total antibodies specific for HMGB1 or the method of monitoringbased on the antibodies specific for HMGB1 mentioned herewith, onsamples obtained from said subject at different times during thetreatment, and determining the efficacy of the treatment given to thesubject.

In this method of the invention, the quantity of antibodies specific forHMGB1 in a HIV infected patient may be compared with the quantity ofantibodies specific for HMGB1 in a non-infected (i.e. non-HIV infected)patient.

Moreover, the quantity of antibodies specific for HMGB1 may be comparedwith the quantity obtained from the same subject at a different time,such as prior to infection, during primary, acute or chronic infection,or prior to the initiation of the treatment, for example before thetreatment and each month during the treatment.

Administration of substance(s) is providing “treatment” according to theinvention, either when the quantity of total antibodies specific forHMGB1 is decreased, preferably from a factor of at least 1.5, at M6 (6months after the initiation of the treatment) or from a factor of atleast 2 or at least 3 at M12, as compared to the quantity of totalantibodies specific for HMGB1 in the same patient before treatment. Theterm “treatment” more generally refers to any means used to reduce theHIV infection, i.e. the HIV RNA and/or the HIV DNA. Treatment accordingto the invention encompasses recourse to conventional treatments usingantiretroviral drugs such as Nucleoside/Nucleotide Reverse TranscriptaseInhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors(NNRTIs), Protease Inhibitors (PIs), Fusion or Entry Inhibitors,Integrase Inhibitors or any combination thereof.

The invention is also directed to an in vitro prognostic method ofeither the state of progression of Acquired immune deficiency syndrome(AIDS) or the state of progression toward AIDS, in a patient infectedwith HIV, comprising carrying out the quantitation method or the methodfor monoriting HIV infection disclosed above in a sample obtained from apatient after infection, and preferably during primary or acuteinfection, or during chronic infection and wherein the more the level ofantibodies specific for HMGB1, the more the risk to develop AIDS or anadvanced state of AIDS.

The term “prognostic” refers to the possibility to evaluate, at the timethe quantitation of the total antibodies specific for HMGB1 is carriedout from a sample obtained from a patient, the risk for the patient todevelop AIDS or to progress toward AIDS. The expression “state ofprogression” refers to the various stages met in the progression of AIDSor toward AIDS, and in particular refers to the WHO Disease StagingSystem for HIV Infection and Disease produced and updated by the WorldHealth Organisation, which is summarized hereinafter. Stage I: HIVdisease is asymptomatic and not categorized as AIDS; Stage II includesminor mucocutaneous manifestations and recurrent upper respiratory tractinfections; Stage III includes unexplained chronic diarrhea for longerthan a month, severe bacterial infections and pulmonary tuberculosis;and Stage IV includes toxoplasmosis of the brain, candidiasis of theesophagus, trachea, bronchi or lungs and Kaposi's sarcoma.

Any of the in vitro methods disclosed above involving the quantitationof the antibodies specific for HMGB1 may be carried out by implementingELISA, or other immunological detection methods, using the High mobilitygroup box I (HMGB1) protein or derivatives thereof coated on a solidsupport, and optionally using secondary antibodies able to detect theHMGB1 specific antibodies.

Based on the results shown below, the inventors have found that HMGB1triggers in vivo HIV replication in HIV-1-infected patients.Consequently, yet another aspect of the invention involves detection ofan increased concentration of HMGB1 in biological samples, such as sera,from HIV-infected subjects. A positive correlation between the viralload and HMGB1 concentration may also be used to monitor HIV infection.Increased HMGB1 levels may be correlated with disease progression orassociated with a worse prognosis. HMGB1 concentration in biologicalsamples may be quantified with well-known diagnostic tests, such asELISA tests. Recombinant hHMGB1, anti-hHMGB1 mAbs and rabbit anti-hHMGB1serum are commercially available and may used in such diagnostic tests.Such a test is used to quantifying HMGB1 concentration in patients'samples, to identify HMGB1 as a prognostic marker of evolution of HIVinfection, and to monitor the in vivo effect of humanized anti-HMGB 1antibodies.

The invention also relates to an in vitro method for monitoring HIVinfection in a subject infected with HIV comprising quantitating Highmobility group box I (HMGB1) protein contained in a biological sampleobtained from said subject, in particular by contacting the biologicalsample from said subject infected with HIV, with antibodies thatimmunologically bind to High mobility group box I (HMGB1), wherein theHMGB1 protein targeted for quantitation is either the total HMGB1protein or its circulating fraction (circulating HMGB1) or itsimmunological complexed fraction.

The methods for monitoring the HIV infection, the viral load or theefficacy of a treatment and the prognostic method disclosed herein, maybe implemented based on the quantitation of the circulating (residual)HMGB1, based on the quantitation of the total HMGB1 or based on thequantitation of the fraction of immunological HMGB1/specific antibodiescomplex.

In a particular embodiment, all these methods are based on either thequantitation of circulating HMGB1 or total HMGB1. The quantitation ofthe total HMGB1 may be preferred when the level of circulating HMGB1 islow. When the quantitation is based on the total HMGB1, the methods ofthe invention also comprise a step suitable for dissociation ofimmunological complexes formed with HMGB1-specific antibodies, and forexample the methods of the invention use or include an acidic treatmentof the sample.

A suitable acidic treatment comprises contacting the sample with anacidic dissociation solution, having a low pH, preferably between pH 1and 3, chosen to separate the HMGB1 protein from the specific antibodywithout altering the HMGB1 protein and its recognition capacity byspecific antibodies. In a particular embodiment, the acidic dissociationsolution is glycine (e.g. 1.5M) at a low pH, preferably between pH 1 and3 (e.g. 1.85). The acid treatment is then stopped with a neutralizationbuffer (such as Tris, for example 1.5M Tris, pH9). In another preferredembodiment, in combination with the previous one or not, the incubationwith the acidic dissociation solution is carried out at a temperaturebetween 20 and 37° C., preferably at 25° C., and/or the neutralizationstep takes place in ice.

The quantitation of the HMGB1 protein may be compared to the amount ofHMGB1 from a biological sample obtained from a subject not infected withHIV, or to the amount of HMGB1 from a biological sample obtained fromthe same subject at a different time.

The invention also concerns a method for monitoring the HIV viral loadin a biological sample obtained from a subject, which is known to beinfected with HIV, comprising carrying out the quantitation of the HMGB1protein, wherein the more the HMGB1 protein, the more the viral load. By“viral load”, it is meant either the HIV RNA (which is derived fromviral particles and present in plasma) or the HIV DNA (which isintegrated in the cell genome and present in cells). In a particularembodiment, the methods of the invention based on the quantitation ofHMGB1 are suitable to monitor the HIV RNA viral load.

The invention also relates to an in vitro method for monitoring theefficacy of a treatment directed against HIV infection in a subjectinfected with HIV, comprising carrying out the method of monitoring theHIV infection based on the HMGB1 protein disclosed above, on samplesobtained from said subject at different times during the treatment, anddetermining the efficacy of the treatment given to the subject, andoptionally comparing these results obtained in a sample of the samesubject prior to the initiation of the treatment. Administration ofsubstance(s) is providing “treatment” according to the invention eitherwhen the quantity of cellular HMGB1 protein is either decreased,preferably from a factor of at least 1.5 at M1 (1 month after theinitiation of the treatment) or from a factor of at least 2 at M3, ascompared to the quantity of HMGB1 in the same patient before treatment,or reached the value obtained in samples of healthy donors (less than500 pg/ml). A treatment may also be considered efficient when thequantity of total HMGB1 protein reached the value obtained in samples ofhealthy donors. The term “treatment” more generally refers to any meansused to reduce the HIV infection, i.e., the HIV RNA and/or the HIV DNA.

The invention also relates to an in vitro prognostic method of eitherthe state of progression of Acquired immune deficiency syndrome (AIDS)or the state of progression toward AIDS, in a patient infected with HIV,comprising quantitating HMGB1 by any method disclosed above, in a sampleobtained from a patient after infection, and preferably during primaryor acute infection, or during chronic infection and wherein the more thelevel of total HMGB1, the more the risk to develop AIDS or an advancedstate of AIDS. The definitions given above, regarding the prognosticmethod based on the antibodies specific for the HMGB1 protein, alsoapply here.

Another aspect of the invention concerns a kit to quantitate the totalantibodies specific for the High mobility group box 1 protein (HMGB1) ina sample, comprising: a) native HMGB1 protein or derivatives thereof asdefined above, and b) an acidic dissociation solution suitable todissociate immunological HMGB1/anti-HMGB1 antibody complexes found inthe sample when taken from the patient, such as defined above.

The invention also relates to a kit to quantitate the total Highmobility group box 1 protein (HMGB1) in a sample, comprising a) anantibody specific for the HMGB1 protein, or a fragment thereof able tobind the HMGB1 protein, as defined above and b) an acidic dissociationsolution suitable to dissociate immunological HMGB1/anti-HMGB1 antibodycomplexes found in the sample when taken from the patient, such asdefined above. Optionally, these kits may also contain a neutralizationbuffer, for example as defined above and/or secondary antibodies bindingto and/or revealing the formation of the HMGB1/specific antibodiescomplex.

Thus, yet another aspect of the invention is the diagnosis, includingdifferential diagnosis, of immunodeficiency virus (HIV) infection or theassessment of the risk of HIV infection in a subject. This diagnosticmethod involves contacting a biological sample, such as blood, plasma,serum, saliva, or other body fluids, obtained from a subject suspectedof being infected with HIV with an antibody that immunologically bindsto High mobility group box 1 protein (HMGB1) and detecting complexformation between any HMGB1 in said sample and the antibody or antibodyfragment that binds to HMGB1. The contact of the sample with saidantibody as well as the detection of the formed complex are carried outin vitro. Diagnosis or an indication of the risk of being infected byHIV may be determined based on increased formation of antibody-HMGB1complexes compared to complex formation in a control subject notinfected with HIV, or compared to complex formation in said subjectprior to HIV infection, such as HIV-1 infection. One example of such adiagnostic method is the use of ELISA to detect HMGB1 protein using amonoclonal antibody coated on a solid support and polyclonal antibody todetect HMGB1 bound to coated mAb.

Other diagnostic tests to exclude or control for acute and/or chronicinflammation in said subject or to indicate the presence or titer of HIVin a test subject may also be performed as part of the overalldiagnostic process of human or non-human subjects.

Similarly, a subject who is known to be infected with HIV or previouslyinfected by HIV may be monitored by contacting a biological sample fromthe subject with an antibody or antibody fragment that binds to Highmobility group box I (HMGB1) protein and quantifying complex formationbetween HMGB1 in said sample and said antibody. The contact of thesample with said antibody as well as the quantification of the formedcomplex are carried out in vitro. Here, the quantity of complexes isindicative of the degree of NK-dependent triggering of HIV replicationin said subject and thus a measure of the severity or progression of HIVinfection. Complex formation may be compared to the amount of complexformation with complex formation in a biological sample obtained from asubject not infected with HIV or with complex formation obtained fromthe same subject at a different time, such as prior to infection orduring a prior acute or chronic infection. This method may also involveother diagnostic tests to exclude or control for acute and/or chronicinflammation attributable of HMGB1 not associated with HIV infection inthe subject and may be accompanied by other diagnostic tests for thepresence of or HIV viral load in the subject.

Prior to performance of a diagnostic assay according to the invention,the biological sample, such as serum or plasma, may be treated with acidto separate HMGB1 from other proteins that bind to HMGB1, see Gaillard,et al., PLOS One 3(8) e2855, pages 1-9 (2008) which is specificallyincorporated by reference as teaching high-sensitivity methods fordetection and measurement of HMGB1 protein, including acid treatment.Such treatment increases the number of HMGB1 epitopes available forrecognition by antibodies. Acid treatment is optional, since someantibodies to HMGB1 bind regions of the protein not blocked by thebinding of other proteins.

Antibodies used for diagnostic applications need not block the activityof HMGB1 and may be polyclonal or monoclonal antibodies or antibodyfragments that bind to HMGB1. Such antibodies may be derived by knownmethods from animals such as mice, rats and rabbits or produced by othermethods well-known in the art. Antibodies or other HMGB1 binding agentsused for diagnostic or monitoring HMGB1 levels may be formulated intokits which include written or electronic instructions regarding how toperform the assay, buffers, preservatives, negative and/or positioncontrol samples, solid supports, and containers or packaging materials.

BRIEF DESCRIPTION OF THE DRAWINGS

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FIG. 1. aNK cells induce the maturation of primary immatureHIV-1-infected DCs. (a) iDCs, generated from purified CD14⁺ monocytes inthe presence of IL-4 and GM-CSF, were cocultured during 24 h with aNKcells at different ratios. DC survival was determined by flow cytometrywith the 7-AAD assay. Surviving DCs were identified as 7AAD⁻CD56⁻ cells.Data represent three independent experiments and values are means±sd.(b) aNK cells induce the maturation of iDCs. Flow cytometry analysis ofiDCs, which were either infected with R5-HIV-1BaL (1 ng/ml of p24) for 3h or uninfected, were incubated with rNK cells or aNK cells at a ratioof 1:5. Co-staining with HLA-DR and CD86 specific antibodies allowed theidentification of mature DCs (CD86^(bright)HLA-DR^(bright)). Data from arepresentative experiment out of three independent experiments areshown. (c) The conditions of infection used in this study were those ofa productive infection of iDCs, as shown at day 3 by a significant p24detection in culture supernatant of infected iDCs and intracellulardetection by flow cytometry of p24 in DC targeted by CD40 expression.Experiments were performed on DCs from three independent donors, andvalues are means±sd (d) HIV-1 infection does not induce by itself thematuration of iDC, as shown by CD86/HLA-DR dual staining of iDCsinfected with 0.001 to 10 ng/ml p24 HIV-1. The proportion of mDCsinduced by LPS (DC0) (78.1% CD86^(bright)HLA-DR^(bright)) is shown as apositive control. (e) The proportion of matureCD86^(bright)HLA-DR^(bright) DCs induced in the indicated cocultures ofinfected or uninfected iDCs with either rNK or aNK cells are shown.These experiments have been performed on primary cells from a number ofdonors, and representative data from three of them are shown. Whenindicated, statistical analyses were made with the non parametricMann-Whitney test. *p<0.05, **p=0.02

FIG. 2. aNK-DC cross-talk triggers HMGB1 expression in both aNK cellsand DCs. (a) 24 h cell-free culture supernatants of iDCs, rNK cells, aNKcells (10⁶/ml), or cocultures of aNK cells and iDCs (ratio 1:5) weretested for cytokine content. MAP technology was used to quantify IL-1β,IL-6, IL-10, TNF-α, IL-12 and IFN-γ, whereas HMGB1 was quantified byELISA. *p<0.05 (non-parametric Mann-Whitney test). (b) HMGB1 expressionwas detected by immunofluorescence (in red) in freshly sorted blood NKcells. Counterstaining with DAPI (in blue) showed the nuclearlocalization of HMGB1. (c) Incubation of aNK cells with HIV-1 inhibitsHMGB1 secretion. Left panel: aNK cells (10⁶ cells/ml) were incubated inmedium or with R5-HIV-1Ba-L (1 ng/ml of p24) for 3 h and tested forHMGB1 production 21 h later. Data represent three independentexperiments and values are means±sd. Right panel: immunofluorescenceanalysis of HMGB1 expression in the same preparations of aNK cells. (d)HMGB1 production during aNK-iDC cross-talk is not inhibited by HIV-1infection of iDCs. iDCs were incubated for 3 h in medium or withHIV-1BaL (1 ng/ml of p24) and further cocultured for 21 h with aNK cells(aNK:iDC ratio 1:5). HMGB1 concentration was then measured in culturesupernatants. Data represent the mean±sd of three independentexperiments. (e) Immunofluorescence confocal analysis of HMGB1expression in uninfected or HIV-1-infected iDCs. Upper panel: noninfected iDCs; middle panel: HIV-1-infected and replicating iDCs, asshown by intracellular p24 staining; lower panel: iDCs incubated withHIV-1 but negative for intracellular p24 expression. (f) Mature DCs weregenerated by 48 h stimulation of iDCs with LPS (DC0), soluble CD40L(DC1) or LPS+PGE2 (DC2). DC0, DC1 and DC2 were incubated for 3 h inmedium or infected with R5-HIV-1 BaL (1 ng/ml of p24) and furtherincubated in medium for 21 h. HMGB1 quantification in culturesupernatants was performed. The mean±sd of three independent experimentsis shown. (g) Immunofluorescence analysis of HMGB1 expression inconjugates of aNK cells and uninfected (upper panel) or HIV-1-infectedDCs (lower panel) in a 24 h coculture. DCs are DC-SIGN⁺ and both aNKcells and DCs express HMGB1 in these conjugates. Pictures from onerepresentative experiment out of three conducted with different primarycell preparations are shown.

FIG. 3. aNK-dependent maturation of HIV-1-infected iDCs is mediated byHMGB1 and involves RAGE. (a) Left panel: iDCs were cultured for 24 heither alone or with aNK cells, in the presence of blocking anti-HMGB1antibodies (10 μg/ml) or glycyrrhizin (10 μg/ml). The maturation statusof DCs was determined by flow cytometry with CD86 and HLA-DR-specificantibodies. Right panel: same experiment, but performed with HIV-1infected iDCs. Data represent mean±sd of at least three independentexperiments, and statistical comparisons were made with the nonparametric Mann-Whitney test. *p<0.05. (b) iDC (10⁶ cells/ml) werecultured for 48 h with increasing concentrations (1-10 μg/ml) ofrh-HMGB1. Cells were then stained with anti-CD86, -HLA-DR, -CD80, -CD83,DC-LAMP and -CD40 antibodies and analyzed by flow cytometry. (c)Influence of rh-HMGB1 on cytokine and chemokine production (determinedby MAP) by DCs. iDCs (10⁶ cells/ml) were incubated for 48 h in medium orin presence of rh-HMGB1 (1 or 10 μg/ml). As a positive control, iDCswere stimulated with LPS (DC0). (d) Flow cytometry detection of surfaceexpression of RAGE by iDCs, DC0, or iDCs incubated with rh-HMGB1 (1μg/ml). iDCs were either non infected or infected with HIV-1 (1 ng/mlp24 for 3 h). (e) iDC, DC0, uninfected or HIV-1-infected iDC coculturedfor 24 h with aNK cells, were incubated with rh-HMGB1 (1 μg/ml) andsubsequently stained with anti-RAGE antibodies and analyzed by flowcytometry. NK cells were excluded from the analysis through theco-staining with CD3- and CD56-specific antibodies (CD3⁻CD56⁺).

FIG. 4. Impairment of NK-triggered Th1 polarization by DCs followingHIV-1 infection is associated to altered IL-12 and IL-18 production. (a)Th1 polarization by DCs triggered by NK cells was tested by incubatingiDC (10⁶/ml) for 30 nm in the presence of rNK or aNK cells (2×10⁵/ml).Naïve CD4 T cells (10⁶/ml) were added to the cocultures and thefrequency of T cells producing IFN-γ or IL-4 was determined by flowcytometry 8 days later. The experiment was performed with eitheruninfected iDCs (b) or HIV-1 infected iDCs (c), or HIV-1 infected iDC inthe presence of AZT (1 mM) (d). Culture supernatants of indicatedcultures were tested for IL-12 (e), IL-18 (f), and IFN-g (g) content.Data represent the mean±sd of five independent experiments. Statisticalcomparisons were made with the nonparametric Mann-Whitney test. *p<0.05,**p=0.03.

FIGS. 5 (a)-(f). HMGB1-dependent triggering of HIV replication in DC asa consequence of NKDC cross talk. (a) Flow cytometry analysis of p24intracellular expression in HIV-1-infected (lower panel) or uninfected(upper panel) iDCs (CD40+) (10⁶/ml) following 3 day-incubation, eitheralone, or in the presence of rNK or aNK cells (2×10⁵/ml). (b) p24concentration in culture supernatants of same cultures. Mean±sd of threeindependent experiments. *p<0.05, non parametric Mann-Whitney test (c)Immunofluorescence analysis of intracellular p24 expression inHIV-1-infected iDCs cultured for 3 days either alone or in the presenceof aNK cells. Nuclei are stained with DAPI. (d) Flow cytometryintracellular p24 expression in HIV-1-infected DC0 (10⁶/ml) culturedeither alone or in the presence of aNK cells for 6 days. (e) p24concentration in culture supernatants of HIV-1-infected mature DCscultured either alone or in the presence of rNK or aNK cells for 6 days.Mean±sd of three independent experiments. Statistical comparisons weremade with the non parametric Mann-Whitney test. *p<0.05. (f) HIV-1proviral DNA levels, determined by light cycle analysis on cells fromindicated cultures. One representative experiment out of three conductedwith different primary cells preparations is shown.

FIGS. 6 (a), (b) and (c). Exogenous rh-HMGB1 triggers HIV-1 and HIV-2replication in iDC. (a) HIV-1-infected iDC were cultured alone or in thepresence of aNK cells for 3 days. Rh-HMGB1 (1 μg/ml) was added in somecultures. HIV replication was measured by p24 quantification in culturesupernatant (b) HIV1-infected iDC were cultured alone or in the presenceof aNK cells for 3 days. Blocking anti-HMGB1 antibodies (10 μg/ml) orglycyrrhizin (10 μg/ml) were added at culture initiation. HIVreplication was measured by p24 quantification in culture supernatant.The mean±sd of three independent experiments is shown. Statisticalcomparisons were made with the non parametric Mann-Whitney test.*p<0.05; (c) HIV-2-infected iDC were cultured alone or in the presenceof aNK cells for 3 days. Rh-HMGB1 (1 μg/ml) was added in some cultures.HIV replication was measured by p24 quantification in culturesupernatant.

FIGS. 7 (a)-(c). Activated NK cells (aNK) rapidly induce apoptosis ofimmature dendritic cells (iDCs) at NK:DC ratio of 5:1. (a) iDCs,generated from purified CD14⁺ monocytes from healthy donors in thepresence of IL-4 and GM-CSF, were co-cultured during 24 h with restingNK cells (rNK) or aNK cells at two different NK:DC ratios (1:5 and 5:1).DCs survival was determined by flow cytometry using the 7-AAD assay. NKcells were excluded from the analysis by gating the CD56⁻ population.Surviving DCs are 7-AAD⁻ FSC^(high) cells. Data represent threeindependent experiments. (b) Live video microscopy of apoptosis of iDCsinduced by aNK cells. Pictures from one representative experiment out ofthree conducted with different primary cell preparations are shown. (c)Kinetics of iDCs killing by aNK cells, assessed by the proportion ofsurviving DCs in cocultures. These experiments have been performed onprimary cells from a number of healthy donors, and representative datafrom three of them are shown.

FIGS. 8( a)-(b). DCs that are resistant to killing by aNK cells exhibita mature phenotype. (a) iDCs, generated from purified CD14⁺ monocytes inthe presence of IL-4 and GM-CSF, were cocultured during 24 h with aNKcells at the NK:DC ratio of 5:1. DCs survival was determined by flowcytometry with the 7-AAD assay. NK cells were excluded from the analysisby gating the CD56⁻ population. Surviving DCs are 7-AAD⁻ FSC^(high)cells. In addition to apoptosis, aNK cells induced the maturation ofiDCs. Co-staining with HLA-DR and CD86 specific antibodies allowed theidentification of mature DCs (CD86^(bright) HLA-DR^(bright)). As apositive control, mature DCs were generated by 48 h stimulation of iDCswith LPS (DC0). Data from a representative experiment out of threeindependent experiments are shown. (b) iDCs cultured alone or coculturedwith aNK cells at the NK:DC ratio of 5:1 were stained with anti-CD83 and-DC-SIGN antibodies and analyzed by flow cytometry. The positiveexpression of both markers demonstrates the mature phenotype of iDCs.Data represent one of three independent experiments.

FIGS. 9( a)-(g). aNK-dependent apoptosis of iDCs is TNF-relatedapoptosis-inducing ligand (TRAIL)-dependent and involves the DR4receptor. (a) CD56⁺ NK cells were purified from the blood of healthydonors. NK cells were maintained in culture with suboptimalconcentrations of interleukin-2 (IL-2) (100 ng/ml) (rNK cells) oractivated (aNK cells) by the addition PHA (10 μg/ml) and IL-2 (10 μg/ml)to cultures. The intensity of staining with anti-CD56 antibodies allowsthe distinction between two NK cell populations expressing CD56 highly(CD56^(bright) cells) and weakly (CD56^(dim) cells). Data represent themean±sd of three independent experiments. (b) Membrane TRAIL (mTRAIL)expression by NK cells is determined by flow cytometry with anti-CD56and -mTRAIL specific antibodies. Data represent one of three independentexperiments. (c) aNK cells were co-stained with anti-CD56 and -mTRAILantibodies. The proportions of mTRAIL-expressing aNK cells amongCD56^(bright) and CD56^(dim) populations were determined. Data representthe mean±sd of three independent experiments. (d) Detection of TRAILreceptor DR4 expression on the surface iDCs by flow cytometry. In somecases, iDCs were cocultured with aNK cells at the NK:DC ratio of 5:1.DR4 expression at the DCs surface is analyzed after 1, 2, 3.5, 6 and 24h of NK-DC coculture. These experiments have been performed on threedonors, and representative data from one of them are shown. (e) iDCs(10⁶ cells/ml) were cultured for 24 h with increasing concentrations(1-1000 ng/ml) of recombinant human soluble TRAIL (rhs-TRAIL). Celldeath was then quantified with the 7-AAD assay. Data represent themean±sd of three independent experiments. (f) 24 h cell-free culturesupernatants of iDCs, rNK cells, aNK cells (10⁶/ml), or cocultures ofaNK cells and iDCs (ratio 5:1) were tested for soluble TRAIL (sTRAIL)content. sTRAIL was quantified by ELISA. Data represent the mean±sd ofthree independent experiments. (g) iDCs were cultured for 24 h eitheralone or with aNK cells (NK:DC ratio of 5:1), in the absence or presenceof blocking anti-DR4 antibodies (250 ng/ml). The viability status of DCswas determined by flow cytometry with the 7-AAD assay. Data representone of three independent experiments.

FIGS. 10( a)-(d). R5-HIV-infected DCs are resistant to killing by aNKcells, although TRAIL secretion persists in the presence of HIV-1 (a)iDCs were either infected with R5-HIV-1_(BaL) (1 ng/ml of p24) for 24 hor uninfected, and after several washes, they were incubated with aNKcells at the ratios of 1:5 and 5:1. iDCs viability was assayed with the7-AAD test by flow cytometry. Living cells are 7-AAA⁻, apoptotic cells7-AAD⁺, and apoptotic debris are 7-AAD⁻ FSC^(low). Dot plots representone of at least three independent experiments. (b) R5-HIV does notinduce iDC maturation. DCs were uninfected (iDCs), infected withR5-HIV-1 at 1 ng/ml of p24 (HIV-DCs) or stimulated with LPS for 48 h.Cells were then stained with CD86 and HLA-DR. Data represent one of atleast three independent experiments. (c) TRAIL secretion is not affectedby HIV infection of iDCs. 24 h cell-free culture supernatants of iDCs,HIV-infected DCs (10⁶/ml), aNK cells-iDCs cocultures (ratio 5:1) and aNKcells-HIV-infected DCs cocultures (ratio 5:1) were tested for solubleTRAIL (sTRAIL) content by ELISA. Data represent the mean±sd of threeindependent experiments. (d) HIV-1-infected DCs are still susceptible toTRAIL-induced apoptosis. iDCs and HIV-infected DCs (10⁶ cells/ml) werecultured for 24 h with increasing concentrations (1-1000 ng/ml) ofrhs-TRAIL. Cell death was then quantified with the 7-AAD assay. Themean±sd of three independent experiments was presented.

FIGS. 11( a)-(b). High-mobility group box 1 (HMGB1) is involved inresistance of HIV-1-infected iDCs to NK-induced DC apoptosis. (a) iDCsor HIV-1-infected iDCs were cultured alone or in the presence of aNKcells (NK:DC ratio of 5:1). In some experiments, azidothymidine (AZT)was added at the time of HIV infection; in others glycyrrhizin (10ng/ml) was added at coculture initiation. Cell death was then quantifiedwith the 7-AAD assay. (b) Same experiments were performed in thepresence of blocking anti-HMGB1 antibodies (10 and 15 μg/ml). Onerepresentative experiment out of three conducted is shown.

FIGS. 12( a)-(b): (a) aNK cells induce apoptosis of uninfected iDCs byupregulating TRAIL receptor (DR4) expression at iDCs surface, increasingthus DC's sensitivity to TRAIL-dependent apoptosis. (b) HIV-1-infectedDCs are resistant to aNK-induced apoptosis by an HMGB1-dependantmechanism. Consequently, aNK cells participate to the persistence ofinfected DC, DC-dependent HIV transmission to CD4 T cells andestablishment of HIV reservoirs.

FIG. 13. Determination of conditions for the ELISA assay for anti-HMGB1antibodies (A): Determination of BSA concentration to saturate wellscoated with HMGB1. (B): Determination of anti-IgG-PAL antibodyconcentration (secondary antibody) to reveal bound anti-HMGB1antibodies. (C): Determination of HMGB1 concentration for coating thewells. (D): Determination of purified anti-HMGB1 antibody concentrationfor elaboration of the standard curve. (E): Specificity of the assay.

FIG. 14. anti-HBG1 titration with coating of the HMGB1 protein or of theBOXB.

FIG. 15. Human sera, either untreated or treated with Glycin 1.5M, weretitrated for the presence of anti-HMGB1 IgG antibodies. Circulating(Free) anti-HMGB1 antibodies were hachured represented, while complexedanti-HMGB1 antibodies were represented in grey.

FIG. 16. titration of HMGB1 concentration in sera from HIV+ patients.Each histogram represents a single patient. The plain line indicates theminimal level of detection by the Elisa test, the dashed lines indicatesthe mean level of HMGB1 in healthy donors.

FIG. 17. Impact of HAART on T cell subsets (A) and HIV viral load (B)measured at different times (in months) following the HAART. ***:p<0.001 and **: p<0.05.

FIG. 18. Titration of HMGB1 in sera from HIV-infected patients receivingHAART at M0. The mean concentration of HMGB1 in healthy donors is shownby the dashed line.

FIG. 19. Titration of anti-HMGB1 antibodies in sera from HIV-infectedpatients, and impact of antiretroviral therapy. M-1 means serum samplesfrom patients tested 15 to 30 days before enrolment in the clinicaltrial. M1, M3, M6 and M12 indicate in the different time pointsfollowing HAART. “Fin” means patients who stopped HAART between M9 andM12.

FIG. 20. Study of correlation between serum HMGB1 and anti-HMGB1antibody concentrations (A), and between anti-HMGB1 antibodies andHIV-RNA viral load (B). Spearman's correlation test. The correlationcoefficient r, probability of correlation (p) and number of samplesanalyzed (n) are indicated.

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EXAMPLES

HMGB1 is a nuclear protein that is present in almost all eukaryoticcells, and it functions to stabilize nucleosome formation and acts as atranscription-factor-like protein that regulates the expression ofseveral genes. It is also a cytokine, secreted by activated macrophages,mature dendritic cells (DCs) and natural killer (NK) cells in responseto injury, infection or other inflammatory stimuli.

Early stages of viral infections are associated with local recruitmentand activation of effectors of innate immunity, i.e. NK cells and DCs.DCs are essential for both antigen-presentation and activation of naïveCD4⁺ T cells, and further Th I polarization. DCs also constitute earlytargets for HIV and contribute to HIV persistence by integratingproviral DNA. DC maturation and homeostasis is controlled by a crosstalkbetween DC and NK cells. The contribution of this cross talk tosusceptibility of DCs to HIV replication was previously unknown.

Activated NK cells (aNK) provide a source of HMGB1, which is releasedduring the contact between NK cells and immature DCs (iDCs) and promotesthe maturation of iDCs and the induction of IL-12-dependent T-helper-1responses. Following infection of iDCs with HIV-1, DCs were no moresusceptible to NK-dependent IL-12 polarization, and thus no more able toinduce a Thl response. In addition, NK-dependent DC maturation andsurvival was associated with an increased production of HIV-1 p24 and anincreased expression of proviral DNA by DCs. NK-dependent increasedreplication of HIV-1 in DCs was inhibited by antibodies specific forHMGB1 and by glycyrrhizin, known to interact specifically with HMGB1,suggesting an important role for this cytokine in this process. As acorollary, rh-HMGB1 had a direct effect on infected DCs, enhancingdramatically the production of p24 in culture supernatants. A strongstimulating effect of HMGB1 on HIV replication in DCs was also observedin aNK: iDC cocultures. The addition of HMGB1-specific neutralizingantibodies or glycyrrhizin abrogated HIV-1 production by infected DCscultivated alone or in the presence of aNK cells. Altogether, theseresults indicate that HMGB1 triggers HIV-1 replication and increasesHIV-DNA in infected DCs, whether added as a recombinant human protein oninfected DCs or produced by aNK cells during NK:DC cross talk.

Although direct infection of DCs is less efficient than infection ofCD4⁺ T cells ^(40, 41) an increasing amount of evidence indicates thatlong-term HIV transmission that is mediated by DCs depends on viralproduction by the DCs^(42, 43, 44), and HIV-infected DCs in vivo mightfunction as viral reservoirs during migration to the lymphoid tissues,thereby helping to spread viral infection.

The inventors have shown for the first time that activated NK cellscontribute to the establishment of viral reservoirs in HIV-1-infectedDCs. The inventors shown herein NK-cell activating capacity ofHIV-1-infected iDCs and the crucial involvement of HMGB1, producedduring aNK-iDC cross-talk, in the stimulation of HIV-1 replication andproviral DNA expression in DCs. A strong impairment of mature infectedDCs to induce Th1 polarization following their cross-talk with NK cellshas also been demonstrated. These observations led to novel therapies toinhibit the ability of HIV to efficiently promote its dissemination andescape the host immune system.

Interaction of NK cells with autologous iDCs results in reciprocalactivation, and this interaction appears crucial in theinitiation/amplification of the early phases of an immune response,before T cells are generated¹¹. NK cells trigger iDCs to mature, andthis occurs through an HMGB1-dependent mechanism²⁰. NK-dependentmaturation of iDCs has been reported to involve a functionalpolarization of DCs, with increase in intracellular free Ca²⁺concentration, cytoskeleton rearrangement, accumulation of secretorylysosomes at the NK/DC synapse, and regulated expression of IL-18 towardthe interacting NK cells. In turn, NK cells secrete large amounts ofHMGB1, which induces maturation of DCs²⁰. The inventors havesubstantiated the involvement of HMGB1 in NK-dependent DC maturationduring NK-DC contact, as shown by the inhibitory effect of anti-HMGB1antibodies or glycyrrhizin, known to interact specifically with HMGB1³¹.Confocal microscopy analyses and HMGB1 detection in cell-free culturesupernatants demonstrated that HMGB1 was not only expressed and secretedby primary NK cells, as reported²⁰, but it was also produced by isolatedDCs, the level of HMGB1 release being linked to their maturation stage.An extremely high level of HMGB1 was detected when iDCs where put incontact with aNK cells, similar to the one released by mature DCs.Interestingly, confocal microscopy analysis of NK-DC conjugates showedthat both cells expressed the cytokine. HMGB1 receptor, RAGE, was foundrapidly induced following DC interaction with aNK cells, and was furtherdown-regulated, compatible with the implication of HMGB1 in NK-dependentDC maturation. In addition to contributing to DC maturation, HMGB1 hasbeen shown to act as a chemoattractant on iDCs⁴⁵, and to be alsorequired for migration of mature DCs in response to CCR7 and CCR4ligands⁴⁶, both activities being mediated by RAGE^(45, 46). Thus HMGB1acts as an alarmin, having activating and chemotactic effects on DCs,and stimulating then the migration of DCs from inflamed tissues to thedraining lymph nodes⁴⁵. These properties of HMGB1 have to be taken intoconsideration in the context of an uncontrolled viral infection, such asthat induced by HIV.

Productive infection of iDCs with HIV-1 preserved NK-dependentphenotypic DC maturation, as shown by the frequency ofCD86^(bright)HLA-DR^(bright) DCs, while HIV itself didn't induce DCmaturation in the range of p24 concentrations used (0.001 to 10 ng/ml).However, the consequence of aNK-DC interaction was a significantenhancement of HIV-1 infection in iDCs. This was shown by several means,indicating an increased frequency of p24⁺ DC, associated with asignificant enhancement of p24 release in NK-DC culture supernatant, andthis was confirmed by immunofluorescence at the single cell level.Moreover, NK-DC cross-talk resulted in a dramatic increase in proviralHIV-1 DNA expression in DCs. Considering the crucial role of HMGB1during the reciprocal activation of DCs and NK cells, its contributionto the triggering of HIV-1 replication in iDCs with blocking anti-HMGB1antibodies or glycyrrhizin was evaluated. The strong blocking effect ofthese inhibitors on p24 release indicates the involvement of HMGB1 inthe process. It is noteworthy that both inhibitors also decreasedsignificantly HIV-1 replication in 24 h cultures of infected iDCs, inthe absence of NK cells. This is likely due to the spontaneous releaseof HMGB1 by iDC, shown here and previously reported⁴⁶, which waspreserved following their infection with HIV-1. These observationsreveal a pivotal role for HMGB1 in controlling HIV-1 replication in DCs.As a corollary, it was demonstrated that rh-HMGB1 significantlyincreased p24 release in culture supernatants of infected DCs and ofaNK-infected DC cocultures. These data may have important implicationsin the understanding of HIV pathogenesis, since plasma HMGB1 levels werefound elevated in chronically HIV-1-infected patients, with the highestconcentrations in patients with clinical complications⁴⁷. Moreover,exogenous HMGB1 was reported to induce in vitro the reactivation ofHIV-1 in PBMCs from HIV-1-infected patients under antiretroviraltherapy³⁹.

Secreted HMGB1 is necessary for proliferation, survival, andpolarization of naïve CD4 T cells after activation by allogeneic DCs,and these effects involve RAGE expressed by DCs⁴⁸. Here, it was shownthat, in syngeneic conditions, HMGB1 was not able by itself to induceTh1 polarization. Indeed, no Th1 response was induced in the presence ofHIV-1-infected DCs, though they continued to produce normal levels ofHMGB1, while being inhibited in the release of IL-12 and IL-18. Recentstudies highlighted the essential role of NK cells in the modulation ofTh1 polarization, suggesting that they trigger IL-12 and IL-18 releaseby DCs, promoting the production of IFN-γ by NK cells that in turntrigger the differentiation of T cells towards Th1 cells^(49, 50). Theessential role of IL-12 and IL-18 on Th1 differentiation is confirmedhere, since the defect of HIV-1 infected DCs to produce increased amountof IL-12 and IL-18 in response to NK cell activation was associated witha defective Th1 polarization. This defect was directly linked to HIV-1replication in DCs, as shown by the positive effect of the HIV inhibitorAZT. These observations suggest that some of the functional alterationsreported in DCs from HIV-infected patients^(51, 52), such as a decreasedsecretion of several cytokines, including IL-12, and an impaired abilityto prime autologous CD4 T cells, may be linked to a defective NK-DCcross-talk, as suggested recently³⁰.

The inventors show that activation of HIV-1 replication and theestablishment of viral reservoirs in HIV-1-infected DCs is dependent ona cross-talk between aNK cells and autologous DCs, and have identifiedthe pivotal role of HMGB1 in this process, produced both by NK cells andDCs during their cross-talk, and showed that NK-dependent triggering ofHIV replication in DCs is completely abrogated by glycyrrhizin, whichbinds specifically to HMGB1, or by blocking with anti-HMGB1 antibodies.In addition, a strong impairment of the ability of HIV-1-infected DCs toinduce Th1 polarization following their cross-talk with NK cells hasbeen demonstrated. Methods of treating and monitoring HIV infection aredescribed based on the role of NK-DC cross-talk in promoting viraldissemination, and on in vivo involvement of HMGB1 in the triggering ofviral replication and replenishment of viral reservoirs.

Example 1 Activated NK Cells Induce the Maturation of Autologous PrimaryImmature Dendritic Cells Infected with HIV-1

The role of NK cells on DC maturation was investigated by generatingmonocyte-derived DCs from isolated monocytes and coculturing them withNK cells purified from the same donor. NK cells were either resting(rNK) or activated by a combination of PHA and IL-2 (aNK). 24 h ofcoculture of aNK cells with autologous immature DC (iDC) induced eitherthe survival or apoptosis of iDCs, dependent on NK-DC ratio, consistentwith previous reports¹⁴. Indeed, aNK-DC ratio of 5:1 induced DCapoptosis, while 1:5 ratio induced DC survival. (FIG. 1 a). iDCssurvival at a NK-DC ratio of 1:5 was associated with their maturation,as shown by the increased coexpression of the maturation markers CD86and HLA-DR (72.1% of CD86^(bright)HLA-DR^(bright) DCs were induced byaNK cells compared to 15.3% at baseline) (FIG. 1 b), a feature of matureDCs. Under the same experimental conditions, rNK cells had a weakereffect on DC maturation, as judged by the proportion ofCD86^(bright)HLA-DR^(bright) DCs (FIGS. 1 b,e). Following infection ofiDC with HIV-1, NK-dependent maturation of iDCs was not altered (FIG. 1b), under conditions of productive infection of iDCs, measured at day 3by p24 release in culture supernatant and intracellular staining of iDCfor p24 (FIG. 1 c).

The direct effect of HIV on DC maturation was found at concentrationsranging from 0.001 to 10 ng/ml, where HIV was unable to increase theexpression of the maturation markers CD86 and HLA-DR, in contrast toLPS, used as a positive control as a strong inducer of DC maturation(FIGS. 1 b,d). Data from three representative donors, shown in FIG. 1 e,confirm the high impact of aNK cells on maturation of iDC after 24 h ofcoculture, whatever the infected or uninfected status of iDC. These datashow that productively HIV1-infected iDCs maintain a normalsusceptibility to maturation induced by NK cells during the NK-DCcross-talk.

Example 2 aNK-Dc Cross-Talk Triggers HMGB1 Expression in Both NK Cellsand DCs

In order to identify the molecules involved in aNK-dependent maturationof iDC, a multianalyte profiling (MAP) was employed to map the keycytokines produced in 24 h culture of iDC, NK cells and aNK:iDC. iDCreleased low amounts of IL-1β, IL-6 and IL-12, and they did not produceIL-10 or TNF-α. Following their coculture with aNK cells, aproinflammatory cytokine profile was induced, with a high increase inIL-12 secretion, significant levels of TNF-α and IFN-γ, both derivedfrom NK cells, and no production of IL-10 (FIG. 2 a). Interestingly,high levels of HMGB1 were detected in those culture supernatants,originating both from iDC and NK cells, and aNK:iDC cocultures resultedin a strong enhancement of HMGB1 concentration in culture supernatants(FIG. 2 a). It was confirmed that at the single cell level, by confocalmicroscopy, that NK cells were able to produce HMGB1, detected in thenucleus of freshly isolated NK cells (FIG. 2 b), and furthertranslocated to the cytoplasm in aNK cells (FIG. 2 c). Following 3 hincubation with HIV-1, aNK cells showed a strong decrease in HMGB1expression, detected both in culture supernatants and by confocalmicroscopy (FIG. 2 c). HMGB1 level reached then a level comparable tothat of rNK cells (FIG. 2 a). The inventors verified that NK cells werenot able to replicate HIV-1, as shown by the lack of p24 detection inculture supernatant and the lack of intracellular p24 staining (detectedby FACS) in NK cells (data not shown). HMGB1 was also secreted by iDCsand, once infected, they still produced comparable amount of thecytokine in culture supernatants (FIG. 2 d). HMGB1 was mostly detectedin the cytoplasm of iDCs, whether infected by HIV-1 or not (FIG. 2 e),and p24 expression in infected DCs did not alter HMGB1 expression, asshown by dual intracellular staining for p24 and HMGB1 (FIG. 2 e). WheniDCs were cocultured with aNK cells, a strong induction of HMGB1secretion in culture supernatants was observed (FIG. 2 d), reachinglevels comparable to those produced by mature DCs, i.e. DC0, DC1 and DC2(FIG. 2 f). Strikingly, HIV-1 infection of iDC did not affect the amountof HMGB1 produced in NK-DC cocultures (FIG. 2 d) and in cultures ofmature DCs (FIG. 2 f). Confocal microscopy analysis showed the formationof conjugates between aNK cells and iDCs, which were also observed whenaNK cells were cocultured with HIV-1-infected DCs, and both cellsexpressed HMGB1, whatever the infected status of DCs (FIG. 2 g). Theseresults demonstrate that HMGB1 is expressed both by NK cells and iDCsduring NK-DC cross-talk, and this process is not altered by HIV-1infection of iDCs.

Example 3 aNK-Dependent Maturation of HIV-1-Infected iDCs is Mediated byHMGB1 and Involves RAGE

To determine the possible involvement of HMGB1 in NK-dependent DCmaturation, glycyrrhizin, which is known to interact specifically withsoluble HMGB1 molecule³¹, was used as well as anti-HMGB1 antibodies(FIG. 3 a). These inhibitors, added at the initiation of the 24 h aNKiDCcoculture, reduced the proportions of mature DCs (identified asCD86^(bright)HLA-DR^(bright)) to the baseline level observed without aNKcells (FIG. 3 a). Similar effect was obtained with infected DCs (FIG. 3a). rh-HMGB1 by itself did not induce phenotypic maturation of iDC, whentreated for 24 h with 1 to 10 μg/ml rh-HMGB1, and similar data wereobtained at 48 h of culture (FIG. 3 b). Indeed, while spontaneousmaturation of iDCs was observed after 48 h of culture in medium, asshown by the high percentage of CD86^(bright)HLA-DR^(bright) DCs, 10μg/ml rh-HMGB1 only weakly increased from 65% to 71% the percentage ofthese cells. Interestingly, rh-HMGB1-treated DCs were not fully mature,as assessed by the lack of expression of CD80, CD83 and the weakexpression of DC-lamp, all fully expressed in mDC (DC0) (FIG. 3 b).However, these partially mature DCs were functionally susceptible torh-HMGB1 as shown by the increased release of the chemokines, MCP1,MIP-1α, MIP-1β and IL-8 by hr-HMGB1-treated DCs (FIG. 3 c). HMGB1receptors include RAGE^(32, 33) TLR-2 and TLR-4³⁴. RAGE was the firstidentified receptor for HMGB1, it is expressed by a variety of immunecells including T cells, monocytes, macrophages and DCs³⁵, and it isused by maturing DCs for in vivo homing to lymph nodes³⁶. While TLR-2and TLR-4 were hardly detected on iDC (not shown), RAGE was fullyexpressed on DCs, as shown by flow-cytometry, and its expression waseven higher on mature DC0 (FIG. 3 d). Following incubation of iDCs with1 μg/ml of HMGB1, down-regulation of RAGE was observed, stronglysuggesting that this receptor was used by these cells (FIG. 3 d).Following DC infection with HIV-1, no change in RAGE levels was detectedon iDC and DC0. Incubation of infected DCs with HMGB1 induced similardown-regulation of RAGE (FIG. 3 d). The possible involvement of RAGEduring NK-DC cross-talk was evaluated with the same approach, comparingRAGE expression on DCs cocultured with aNK cells and DC cultured alone.After 2 h of coculture with aNK cells, DCs showed an up-regulation ofRAGE expression, followed by a down-regulation at 24 h (FIG. 3 e). Verysimilar observations were made with HIV-1-infected DCs (FIG. 3 e). Thus,HMGB1 is an important factor for the maturation of both uninfected andHIV-1-infected iDCs during NK-DC cross talk, and it involves RAGE, whoseexpression on iDC is not altered following their productive infection.

Example 4 Impairment of Th1 Polarization by HIV-Infected DCs as aConsequence of a Defective NK-DC Cross-Talk

The interaction of NK cells with iDCs results in the induction of type-1polarized DCs that serve as carriers of the NK cell-derived help for theinduction of Th1 responses³⁷. To assess the capacity of DCs, whetherinfected or uninfected, to polarize a Th1 response following theircross-talk with aNK cells, naïve CD4⁺CD45RO⁻T cells were cocultured for8 days in the presence of DCs and aNK cells, and Th1 polarization wasdetermined by the detection in T cells of the intracellular productionof IFN-γ and IL-4, measured by FACS (FIG. 4 a). Coculture of naïve Tcells with iDCs did not increase the proportion of IFN-γ positive Tcells, and similar data were obtained in coculture of naïve T cells withiDCs and rNK cells. In contrast, cocultures of naïve T cells with iDC inthe presence of aNK cells induced a significant increase of IFN-γ T cellresponse (FIG. 4 b), suggesting that aNK:iDC cross-talk is essential forTh1 polarization. When the same experiment was performed withHIV-1-infected DC, no Th1 polarization was observed (FIG. 4 c). Thecontribution of HIV-1 replication to the inhibition of Th1 polarizationwas shown by the addition of AZT, which restored the increased IFN-γ Tcell response induced by infected DCs cocultured with aNK cells (FIG. 4d). AZT was used at a concentration inhibiting viral replication inthese conditions, as assessed by the dosage of p24 antigen in thesupernatants (data not shown). IL-12 and IL-18 are critical cytokinesproduced by DCs and involved in Th1 polarization. This addressed thequestion of the impact of aNK-DC cross-talk on the release of thesecytokines by DCs. It was found that aNK-DC cross talk triggers bothIL-12 and IL-18 secretion by non infected DCs. Importantly, theproduction of both cytokines was not detected anymore in cocultures ofaNK cells and infected DCs (FIG. 4 e, f). In addition, the triggering ofIFN-γ production by NK cells during aNK-DC cross talk was not detectedanymore when the coculture was performed with HIV-1 infected DC (FIG. 4g). Thus, the priming of DCs for Th1 polarization occurs during aNK-iDCcross-talk, though the induction of cytokines such as IL-12 and IL-18released by DCs, and IFN-γ released by NK cells. Following theirinfection with HIV-1, iDCs cannot be polarized anymore by aNK cells, dueto a defective NK-DC cross-talk. Consequently HIV-1 infected DCs areimpaired in their capacity to induce Th1 polarization.

Example 5 Pivotal Role of HMGB1 in Nk-Dc Dependent Triggering of HIV-1Replication and Persistence in iDCs

Since it was shown that the impairment of Th1 polarization byNK-sensitized HIV-1 infected DCs was dependent on HIV-1 replication(FIG. 4 d), the inventors tested whether aNK-iDC interaction couldtrigger HIV-1 replication in iDCs. iDCs were infected for 3 h with HIV-1(1 ng/ml of p24) and further cultured either alone or in the presence ofrNK or aNK for 18 h, and the frequency of DCs with intracellularexpression of p24 was determined by flow cytometry. While the percentageof p24⁺ DCs was quite low when infected iDCs were cultured alone, itsignificantly increased following their interaction with aNK cells, thep24⁺DCs representing almost one third of all DCs as compared to only 4%in the absence of NK cells (FIG. 5 a). Under the same conditions, rNKcells had no effect on HIV replication in iDCs (FIG. 5 a). aNK-dependentincreased HIV replication in infected DCs was confirmed by p24 antigendetection in culture supernatants, and a statistically significantincrease of p24 production was detected in cocultures of aNK withHIV-1-iDC as compared to infected iDCs cultured alone or with rNK cells(FIG. 5 b). The dramatic effect of NK-DC interaction on the frequency ofp24-expressing DCs was confirmed by confocal microscopy withp24-specific antibodies. While very rare DCs were stained forintracellular p24 on the day following their infection, a high number ofp24⁺ DC were observed after their culture with aNK cells (FIG. 5 c).Interestingly, the positive influence of aNK cells on HIV replication iniDCs was similarly observed on mature DCs. An increased frequency ofp24⁺DCs, detected by FACS, was found in HIV-1-infected DC0 coculturedduring 24 h with aNK cells as compared to DC0 cultured alone (FIG. 5 d),and p24 detection in culture supernatants from HIV-1-infected matureDC0, DC1 and DC2, cocultured with aNK cells, confirmed the significantstimulating effect of aNK cells on HIV-1 replication in mature DCs (FIG.5 e). Of note, rNK cells had no significant impact on HIV-1 replicationin mature infected-DCs (FIG. 5 e). The inventors then tested whether aNKcells had an influence on the expression of proviral DNA in iDCs. Datain FIG. 5( f) show that a very high increase in the number of HIV-1proviral DNA copies was detected in cultures of infected iDCs with aNKcells, as compared with that of infected iDCs with rNK cells or infectediDCs alone.

Exogenous HMGB1 was recently reported to increase HIV-1 replication ininfected monocytic cell lines³⁸, and to induce in vitro the reactivationof HIV-1 in PBMCs from HIV-1-infected patients under antiretroviraltherapy³⁹. Therefore, the question of the role of HMGB1 in theNK-dependent triggering of HIV replication in DCs was addressed. It wasfound that exogenous rh-HMGB1 had a direct effect on HIV-1-infected iDC,enhancing dramatically the production of p24 in culture supernatants(FIG. 6 a). rh-HMGB1 had also a significant stimulatory effect on p24production by HIV-1-infected iDC cocultured with a NK cells (FIG. 6 a).To investigate the influence of HMGB1 in the triggering of HIV-1replication in infected-iDC-aNK cocultures, HMGB1-specific neutralizingantibodies or glycyrrhizin were added to these cocultures and p24production was measured in the supernatant. Both HMGB1 inhibitorsabrogated HIV-1 production by infected DC cocultured with aNK cells orcultured alone (FIG. 6 b). These results indicate that exogenous HMGB1is able to trigger HIV-1 replication by infected iDC. They also indicatethat aNK cell-dependent stimulation of HIV-1 replication in iDCs ismediated by HMGB1.

Example 6 Isolation and Separation of Primary Cells

Peripheral blood mononuclear cells (PBMCs) were separated from the bloodof healthy donors (EFS Cabanel, Paris, France) on a Ficoll-Hypaquedensity gradient. CD14⁺ monocytes were isolated from PBMCs by positiveselection using CD14-specific immunomagnetic beads (Miltenyi Biotech,Auburn, Calif.). To generate iDCs, purified CD14⁺ monocytes werecultured for 6 days (1×10⁶ cells/ml) in RPMI 1640 medium supplementedwith 2 mM glutamine, 10% FCS, penicillin (100 U/ml) and streptomycin(100 μg/ml), in the presence of 10 ng/ml of recombinant human (rhu)GM-CSF and 10 ng/ml rhIL-4 (Peprotech INC, Rockyhill, USA) asdescribed⁵³. Culture medium was replaced every 2 days. NK cells wereisolated by negative selection from PBMCs depleted of monocytes using adepletion cocktail of antibodies directed to CD3, CD4, CD14, CD19, CD20,CD36, CD123, CD66b, Glycophorin A (StemCell Technologies). The NK cellcontent of the enriched fraction, determined by flow cytometry(FACScalibur, Becton Dickinson) as CD3⁻CD56⁺ cells with FITC-conjugatedanti-CD3 and APC-conjugated anti-CD56 antibodies, ranged from 85 to 95%in the different experiments. Contamination with myeloid cells,evaluated with FITC-conjugated anti-CD14 antibodies was consistentlyless than 1%. Naïve CD4 T cells (CD4⁺CD45RA⁺) were isolated from PBMCsby positive selection, using CD4- and CD45RA-specific immunomagneticbeads (Miltenyi Biotech, Auburn, Calif.). Cell purity of isolated naïveCD4 T cells was routinely more than 90%.

Example 7 Activation and Infection of NK Cells

Purified NK cells were cultured at 10⁶ cells/ml either in the presenceof suboptimal concentration of IL-2 (100 ng/ml) (Peprotech) to maintainthem alive (referred as rNK) or were activated by a combination of PHA(10 μg/ml) (Sigma) and IL-2 (10 μg/ml) (referred as aNK cells). In someexperiments, aNK cells (10⁶ cells/ml) were incubated during 3 h in thepresence of HIV-1 (1 ng/ml p24) and further cultured for 21 h. Underthose conditions, no productive infection could be observed. Culturesupernatants were then tested for cytokine and chemokine detection (seebelow).

Example 8 Maturation and Phenotypic Analysis of Dendritic Cells

After 6 days of culture in the presence of IL-4 and GM-CSF, iDCs (10⁶cells/ml) were either non stimulated, or stimulated during 48 h with 10μg/ml LPS (E. coli serotype 026-B6, Sigma-Aldrich) to obtain DC0 cells,or 500 ng/ml of trimeric CD40L (Sigma-Aldrich) to obtain DC1 cells, or10 μg/ml of LPS and 1 μg/ml PGE2 (Sigma-Aldrich) to obtain DC2 cells.Phenotypic analysis of DCs and characterization of their maturationstage was performed by flow cytometry. DCs were stained for 20 min at 4°C. with antibodies specific for CD80, CD83, CD86, HLA-DR, CD40, DC-LAMPor DC-SIGN (all antibodies from BD Biosciences, San Jose, Calif.)diluted in 100 μl of PBS/10% FCS/0.1% NaN³. In some experiments,antibody specific for HMGB1-receptor, RAGE (Abcam), was used to stainDCs. After two washings, cells were fixed in 1% PFA, immediatelyacquired on a FACScalibur (Becton Dickinson) and analyzed with Flow Josoftware.

Example 9 Infection of Dendritic Cells with HIV-1

Virus stock preparation was prepared by amplification of R5-HIV-1Ba-L onMDM from healthy donors. Viral stock was then clarified bycentrifugation prior to determination of HIV1 p24 concentration. iDCswere plated in 96-well culture plates at 200,000 cells/well andincubated for 3 hours at 37° C. in a 5% CO2 atmosphere with R5-HIV-1BALat various concentrations (0.001 to 10 ng p24/ml). Cells were harvested,washed four times with media containing 10% FCS and, when indicated, rNKor aNK cells were added at a NK:DC ratio of 1:5, unless otherwiseindicated. NK-DC cocultures lasted 24 h before analysis of thematuration stage of DCs and/or quantification of viral production. Insome experiments (FIG. 6), HIV-1 infected iDCs were incubated alone orwith aNK cells for 3 days, in the presence of rh-HMGB1 (1 μg/ml)(R&DSystems), and in some cultures in the presence of rabbit antiHMGB1Abs (10 μg/ml) (Abcam, Cambridge, UK) or Glycyrrhizin (10 μg/ml).

Example 10 Quantification of HIV-1 Viral Production, Proviral Load andof the Frequency of Infected DCs

The concentration of HIV-1 in the supernatant of infected cell cultureswas determined by measuring the amount of p24 protein by ELISA (Ingen,Belgium). DNA from cells was extracted using the GIAamp DNA Blood MiniKit (Qiagen, Basel, Switzerland) and quantified HIV-1 proviral load byRT-PCR as described previously⁵⁴. The frequency of HIV-1-infected cellswas determined by flow cytometry to detect intracellular p24 molecule.Cells were surface stained with antibodies specific for CD40 (BDBiosciences, San Jose, Calif.) to target DC and intracellular stainedwith p24-specific antibodies (Coulter). Stained cells were fixed in 1%PFA, immediately acquired on a FACScalibur (Becton Dickinson) andanalyzed with FlowJo software. In some experiments infected DCs wereimaged, after immunofluorescence, by laser scanning confocal microscopy.

Example 11 Cocultures of iDCs with NK Cells

rNK or aNK were cocultured during 24 h with iDCs or mDCs at a ratio of1:5 (2×10⁵ NK+10×10⁵ DC/1 ml), unless otherwise indicated. DC survivalwas determined with the 7-AAD assay, as described previously⁵⁵. Briefly,cultured cells were stained with 20 μg/mL nuclear dye7-amino-actinomycin D (7-AAD; St. Quentin-Fallavier, Sigma-Aldrich) for30 minutes at 4° C., and co-stained with CD56-specific antibody (BDBiosciences, San Jose, Calif.). Surviving DC were identified asCD56⁻7-AAD⁻ cells. When phenotypic characterization of DCs was performedin NK-DC cocultures, NK cells were always excluded from the FACSanalysis through their staining with CD56-specific antibodies.

Example 12 Measurement of Cytokine and Chemokine Production

Cell-free culture supernatants were prepared by incubating for 24 h iDCsat 10⁶ cells/ml, rNK or aNK cells at 2.10⁵ cells/ml or aNK and iDC cellsat the ration of 1:5. Chemokines and cytokines were measured by Luminex(24 plex kits; Biosource) following the manufacturer's instructions. Inbrief, 50 μl of supernatant or standard was incubated withantibody-linked beads for 2 h, washed twice with wash solution, andincubated for 1 h with biotinylated secondary antibodies. A finalincubation of 30 min with streptavidin-PE preceded the acquisition onthe Luminex 100IS. At least 100 events were acquired for each analyte.Values above or below the standard curves were replaced by the lowest orthe highest concentrations measured. Quantification of HMGB1 in cellfree culture supernatants was performed with an ELISA kit (IBL,Hamburg). In experiments testing Th1 polarization of naïve CD4 T cellsby NK-triggered DCs (FIG. 4), quantification of IL-12, IFN-γ and IL-18in culture supernatants was performed with ELISA kits (IL-12 and IFN-γkits from R&D Systems, IL-18 kit from MBL).

Example 13 Th1 Polarization Assay

Naïve CD4 T cells (10⁶/ml) were cocultured for 8 days in the presence ofuninfected or HIV-1-infected iDC (10⁶/ml) and resting or activated NKcells (2×10⁵/ml) and tested for Th1 polarization by flow cytometry, aspreviously reported⁵⁶. Briefly, brefeldine A (10 μg/ml) (Sigma Aldrich)was added during the last 16 h of the culture to inhibit proteinsecretion. Surface staining was performed with PerCP-conjugated CD8antibodies and FITC-conjugated CD3 antibodies (BD Biosciences, San Jose,Calif.), followed by cell fixation for 15 minutes at 4° C. with 1% PFAand permeabilization with saponin buffer (PBS-BSA 0.2% —NaN³ 0.01%-saponin 0.5%), and intracellular staining was performed withAPC-conjugated IFNγ- or IL-4-specific antibodies (BD Biosciences, SanJose, Calif.). Stained cells were immediately acquired on a FACScalibur(Becton Dickinson) and analyzed with Flow Jo software. In order toanalyze the influence of HIV-1 replication on Th1 polarization, AZT 1 mMwas added at the initiation of the culture of naïve CD4 T cellsincubated alone, or in the presence of HIV-1 infected iDCs +/−rNK or aNKcells. AZT was left until the end of the coculture. HIV-1-infection ofiDCs was performed as described above, in the absence of AZT.

Example 14 Statistical Analysis

Statistical analyses were made with the non parametric Mann-Whitneytest. The P value of significant differences is reported. Plotted datarepresent mean±standard deviation (s.d.).

Example 15 HIV-1-Infected Dendritic Cells are Resistant to NK-InducedApoptosis Through an HMGB1-Dependent Mechanism

Dendritic cells (DCs) and natural killer (NK) cells are key innateeffectors playing a critical role in early defenses against infections.Evidence of an NK-DC crosstalk has emerged recently. This crosstalk isbidirectional and it may lead to both NK cell activation anddifferentiation into killer cells, DC maturation or apoptosis, dependingon the activation state of both cell types. DCs are required for thepriming of helper CD4⁺ T cells into Th1 effectors, and the chronicexpression of uncontrolled viruses, such as HIV, may induce impairedmaturation and destruction of DCs. In this study, we addressed thequestion of the impact of NK-DC interaction on the destruction of DCs,and the influence of HIV on this crosstalk.

Immature DCs (iDCs) were prepared from sorted monocytes from healthydonors, cultured for 6 days in the presence of IL-4 and GM-CSF. In someexperiments, iDCs were infected with R5-HIV-1 (1 ng/ml of p24).Coculture experiments with autologous purified aNK cells (activated byPHA+IL-2) were performed at various NK: DC ratios. The influence ofNK-DC interaction on DC's maturation and apoptosis was analyzed usingmultiparametric flow cytometry, combining 7-AAD staining with membraneand intracellular staining with mAbs specific for HLA-DR, DC-SIGN, CD83,CD86, DR4, mTRAIL, etc.

It was found that aNK cells rapidly (within 1-2 h) induce apoptosis ofuninfected iDCs at the NK:DC ratio of 5:1. Live videomicroscopy of NK-DCcocultures confirmed that directly after an NK-DC contact, DCs show atypical apoptotic phenotype (increase in cell's volume and bubbling)(FIG. 7). Surviving DCs exhibit the phenotype of mature cells (FIG. 8).iDC apoptosis involves TNF-related apoptosis induced ligand (TRAIL)produced by aNK cells, and it is mediated by the interaction betweenCD56^(bright) NK cells expressing TRAIL at their membrane level and DCsexpressing the TRAIL's receptor DR4. NK-dependent iDCs apoptosis iscompletely abrogated by neutralizing anti-DR4 antibodies, highlightingthe important role of the TRAIL-dependent pathway in this process (FIG.9). However, the addition of Concanamycin A (an inhibitor of granules'dependant cytotoxicity) to NK-DC cocultures has no effect onNK-dependent DCs apoptosis, excluding the implication of the perforinpathway in DC apoptosis.

To investigate the impact of HIV on aNK-induced iDCs' apoptosis, iDCswere infected with R5-HIV-1 (1 ng of p24/ml). Following HIV infection,iDCs became resistant to aNK-dependent apoptosis. HIV-1 did not induceby itself the maturation of iDCs, as shown by CD86/HLA-DR co-staining ofinfected iDCs (FIG. 10). TRAIL secretion by aNK cells was not affectedby HIV infection of iDCs, and HIV-1-infected DCs were still susceptibleto TRAIL-induced apoptosis (FIG. 10). Resistance of HIV-1-infected iDCsto aNK-induced apoptosis was found dependent on HMGB1, as shown byinhibition assays in the presence of glycyrrhizin or blocking anti-HMGB1antibodies (FIG. 11). Resistance of HIV-1-infected iDCs to aNK-inducedapoptosis was also found dependent on HIV-1 replication in DCs, asdemonstrated by the addition of azidothymidine (AZT) at the time of iDCinfection (FIG. 11). Altogether, these results show that HIV-1 infectionof iDCs induces resistance of infected iDCs to aNK-induced apoptosis,involving the proinflammatory cytokine HMGB1.

The inventors previously recognized that the cross-talk between aNKcells and HIV-1-infected iDCs resulted in a dramatic increase in viralreplication and proviral DNA expression in DCs, and this process wasmainly triggered by HMGB1. The results in this Example show the criticalinvolvement of HMGB1 as a key mediator in the survival of HIV-1infected-DCs, highlighting then the role of HMGB1 in viral persistenceand establishment of HIV reservoirs. These results show how HIV‘hijacks’ DCs to promote efficiently viral dissemination and how thisproperty can be used to treat HIV infection.

Example 16 aNK-DC Cocultures Experiments in the Presence of HIV-2

The question of the susceptibility of HIV-2-infected DCs on NK-dependenttriggering of viral replication was addressed, similarly to HIV-1. Theinfluence of HMGB1 in that process was evaluated.

1—Infection of DCs with HIV-2: iDCs were plated in 96-well cultureplates at 500,000 cells/well and incubated for 3 hours at 37° C. in a 5%CO2 atmosphere with HIV-2 (20 ng p24/ml).

2—NK-DC cocultures: cells were harvested, washed three times with RPMIcontaining 10% FCS and, when indicated, aNK cells were added at a NK:DCratio of 1:5. When indicated, recombinant HMGB1 was added at 10 μg/ml(R&D Systems), or rabbit anti-HMGB1 Abs (1 μg/ml) (Abcam, Cambridge,UK). NK-DC cocultures lasted 3 to 7 days before quantification of viralproduction in culture supernatants.

3—Quantification of HIV-2 viral production: the concentration of HIV-2particles in the supernatants was determined with the p24 ELISA kit(Ingen, Belgium).

As shown on FIG. 6C, a very low level of HIV-2 production was detectedafter three days of infection, whether infected DCs were cultured aloneor in the presence of aNK cells. rh-HMGB1 induced a slight increase inviral replication. As observed in our previous studies with HIV-1, day 3of infection of DCS is too early to detect significant viralreplication. These coculture supernatants will be tested again at day 7.

Example 17 Detection of HMGB1 Protein and Anti-HMGB1 Antibodies in HumanSera/Association with Disease Activity in Patients Infected with HIV

The concentration of HMGB1 protein in sera from HIV-infected patientswas quantitated, according to the ELISA kit Shino Test (IBL).

Moreover, a specific Elisa assay for the detection of totalanti-HMGB1-specific antibodies was develop. Considering thatautoantibodies specific for HMGB1 can be found in SLE (Systemic lupuserythematosus) (Hayashi et al., 2009), it was asked whether anti-HMGB1antibodies were detected in HIV-infected patients and if their levelswere correlated with HIV infection.

Elisa Assay for the Detection of Anti-HMGB1 Antibodies

The assay was developed in two steps:

(1) In a first step, rabbit polyclonal antibodies specific for humanHMGB1 were used, to define the conditions for titration of antibodies oncoated HMGB1 or BOX B. Since anti-HMGB1 antibodies were suspected to befound as immune complexes, in serums (Urbonaviciute et al. Factorsmasking HMGB1 in human serum and plasma. J Leukoc Biol. 2007 81:67-74),a method to dissociate these complexes before titration of antibodieswas develop.

(2) In a second step, human samples from several groups of donors (serafrom either healthy donors, septic choc patients or HIV⁺ patients beforeand after antiretroviral treatment) were used.

The following Reagents were used:

Rabbit primary polyclonal antibodies to human HMGB1 (Adcam ab18256) aredirected against a KLH-conjugated synthetic peptide derived fromresidues 150 to C-terminus of human HMGB1.

Recombinant HMGB1 (HMGBiotech, HM-115) produced in E. Coli from anexpression plasmid coding for rat HMGB1, 99% identical to the humanHMGB1.

Recombinant BOXB from HMGB1 (HMGBiotech HM-051) produced in E. Coli froman expression plasmid coding for the mammalian sequence, which istotally identical in human and mouse.

Control rabbit serum (Sigma; Ref: R9133)

anti-rabbit IgG or IgM conjugated to phosphatase alkaline (PAL),substrate p-nitrophenyl phosphate tablets (pNPP),

calibrators: human IgG from serum (Sigma; ref 12511) and Human IgM fromserum (Sigma; ref 18260)

Anti-human IgG (Fc specific)-alkaline phosphatase antibody produced ingoat (Sigma; Ref A9544), anti-human IgM (μ-chain specific)-alkalinephosphatase antibody produced in goat (Sigma; ref A3437)

The following assay was carried out:

Coating of 96-well plates was performed overnight at 4° C. with either 3μg/ml of HMGB1 or 0.5 μg/ml of BOXB in DPBS. Simultaneously, coating ofthe calibrator was performed with serial dilutions in DPBS ofcorresponding isotypes (only for ELISA assay carried out with humansamples). Plates were washed four times with DPBS/0.05% (v/v) Tween® 20,using a microplate washer (Atlantis; Oasys). Similar washings wereperformed after each step of the ELISA assay. Unbound sites were blockedat 4° C. for 2 hours with PBS/2% (w/v) BSA. 100 μl aliquots of serumsample diluted in DPBS/0.05% (v/v) Tween®/1% (W/V) BSA were added tocoated and uncoated wells and incubated for 1 hour at 37° C. All serumsamples have been tested either untreated or treated with 1.5M Glycine(v/v, pH 1.85) for 30 nm at 25° C. in a water bath, and further kept onice and diluted with 1.5M Tris, v/v, pH 9.0. Samples were thenimmediately diluted (from 1/10 to 1/1000) and distributed on coatedplates. Anti-rabbit IgG alkaline phosphatase-conjugated antibodies(ratio 1/10000), or goat anti-human IgG (ratio 1/2000), or IgM (ratio1/2000) alkaline phosphatase-conjugated antibodies diluted in DPBS/0.05%(v/v) Tween®/1% (W/V) BSA were added for 1 hour at 37° C. Detection ofantigen-specific antibodies was performed after 30 nm of incubation at37° C. with 100 μl pNPP substrate and the reaction was stopped byaddition of 100 μl NaOH 3M. Concentration of HMGB1- or BOXB-specificantibodies has been calculated according to the standard curve obtainedfrom standard immunoglobulin solution absorbance by Ascent software,ThermoElectrocorp, as we previously reported in an Elisa specific forShigella LPS (Launay et al. Vaccine 2009, 27:1184-1191). The data areexpressed in μg/ml of antibodies detected.

To develop this assay, a number of parameters have been tested, usingeither HMGB1 or BOXB coated plates to titrate rabbit anti-humanantibodies. The results obtained are the following:

-   -   a 2% to 5% BSA concentration is equally efficient (FIG. 13 A);    -   a 1/10 000 dilution of the anti-IgG-PAL antibody has been chosen        as being in the linear part of the titration curve for purified        anti-HMGB1 antibodies, shown by the arrows (FIG. 13B);    -   concentrations from 2.5 to 5 μg/ml of HMGB1 for coating the        wells were the most appropriate as shown by the linearity of the        titration curves for purified anti-HMGB1 antibodies (FIG. 13C);    -   a concentration of purified anti-HMGB1 antibody of 0.5 μg/ml was        chosen (FIG. 13D);    -   the test was specific since there is no reactivity of non immune        rabbit antibodies as compared with rabbit anti-HMGB1 antibodies        (FIG. 13E); and    -   comparable data were obtained when purified rabbit anti-human        antibodies were tested on either HMGB1 or Box B-coated plates.        Box B (the main immunogenic part of HMGB1) was further chosen        (FIG. 14).

Acidic Treatment for the Detection of Complexed Anti-HMGB1 Antibodies inHuman Samples

To determine the assay conditions required for testing human biologicalsamples, a series of human sera have been titrated for the presence ofHMGB1-specific antibodies, and assuming that [HMGB1 anti-HMGB1 Ab]complexes were present in biological samples, the influence ofpretreatment with Glycine 1.5M, pH1.85 to dissociate these immunecomplexes has been analyzed. All serum samples have been tested eitherbeing untreated or treated with 1.5M Glycine (v/v, pH 1.85) for 30 nm at25° C. in a water bath, and further kept on ice and diluted with 1.5MTris, v/v, pH 9.0. Samples were then immediately diluted and distributedon coated plates and tested as described above.

The data presented in FIG. 15 show that anti-HMGB1 antibodies werehardly detected in human sera, unless they were treated with Glycin 1.5Mto dissociate the immune complexes. Thus, most of the HMGB1-specificantibodies formed complexes with HMGB1, representing a neutralizationmechanism for proinflammatory molecules.

Quantification of HMGB1 and Anti-HMGB1 Antibodies in Sera from HIVPatients

Circulating HMGB1 and anti-HMGB1 antibodies have been tested inuntreated HIV-infected (HIV⁺) patients at different stages of thedisease.

1. HMGB1 Titration in Sera from HIV-Infected Patients

FIG. 16 shows that increased circulating levels of HMGB1 are detected inHIV+patients as compared to healthy donors (dashed line).

2. Impact of Potent Antiretroviral Therapy on CD4 Cells, CD8 Cells,Proviral DNA and HIV RNA VL

In the course of a one-year clinical follow-up of seven HIV+ patients(with detectable viral load; VL), the immunological effect of a highlyactive antiretroviral therapy (HAART) composed of a combination ofanti-HIV drugs (blocking HIV entry and replication into the host cell),CD4 cells, CD8 cells, proviral DNA and HIV RNA VL were measured atinitiation of HAART (M0), and after 1 (M1), 3 (M3), 6 (M6) and 12 (M12)months of HAART. Results are presented in the following Table and inFIG. 17.

PATIENTS P1 P2 P3 P4 P5 P6 P7 M0 HIV-RNA 6.72 4.15 3.54 5.65 4.90 6.153.85 (log₁₀ cp/ml) M1 Δ HIV-RNA 3.72 2.37 1.50 3.51 2.09 2.35 1.46(log₁₀ cp/ml) M3 Δ HIV-RNA 3.25 2.85 1.94 3.40 2.32 2.10 2.24 (log₁₀cp/ml) M6 Δ HIV-RNA 4.82 2.85 1.40 3.96 2.30 0.81 2.24 (log₁₀ cp/ml) M12Δ HIV-RNA 4.84 2.85 2.24 2.88 3.30 2.21 2.55 (log₁₀ cp/ml)

This table of patients' characteristics show that HAART induces asignificant and rapid suppression of HIV-RNA VL in all the patients (Δviral load means the difference between VL at a given time point and VLat baseline M0), reaching undetectable levels (50 copies/ml blood).Moreover, HAART induces a significant increase in the number of bloodCD4 T cells, while no change was detected at the CD8 T cell level (FIG.17A). HAART also induces a significant decrease in plasmatic HIV-RNAviral load (p<0.001 at M1, M3 and p<0.05 at M6 and M12 vs M0. Nosignificant effect on cell associated HIV-DNA was observed (FIG. 17B).

3. Impact of Potent Antiretroviral Therapy on HMGB1 and Anti-HMGB1Antibodies in these Serum Samples.

Plasma levels of HMGB1 and anti-HMGB1 antibodies were titrated atinitiation of HAART (M0), and after 1 (M1), 3 (M3), 6 (M6) and 12 (M12)months of HAART. Antibody titers have been determined by the assaydescribed above, i.e. that patients' sera have been treated with Glycinebefore titration, and quantitated for anti-HMGB1 antibodies. Results arepresented in FIGS. 18 and 19.

Titration of HMGB1 in serum samples from these patients showed thatsuppression of HIV-RNA VL under HAART was associated with decreasedlevels of HMGB1 (FIG. 18). By M6, HMGB1 levels reached those of healthyindividuals (dashed line). Thus, the impact of HAART argues for adriving role of HIV upon HMGB1 production.

Data in FIG. 19 show that detectable concentrations of anti-HMGB1antibodies were found in patients' sera at M0, and antiretroviraltherapy induced a drop in anti-HMGB1 concentration by M6, reachingundetectable values at M12. Statistically significant decrease inanti-HMGB1 antibody levels as compared to baseline was detected at M6(p=0.05) and at later time points (M12) as well.

Therefore, the combined measures of HMGB1 and anti-HMGB1 levels indicatethat chronic HIV infection triggers the production of HMGB1, which inturn triggers the production of neutralizing antibodies. This is adynamic process implying a delay between a drop of HMGB1 levels (M3) anda decrease of anti-HMGB1 levels (M6), the levels of both molecules beingnormalized following potent antiretroviral therapy.

Correlations Between Anti-HMGB1 Levels and HIV Viral Load

Considering the above-mentioned results, an important question wasaddressed regarding whether circulating levels of HMGB1 and anti-HMGB1antibodies were correlated with HIV-RNA viral load.

Therefore, serum samples from HIV+ patients were tested, at differentstages of the disease, with variable viral load. The results aresummarized in the following table (Spearman's correlation test), and inFIG. 20.

HMGB1 HIV-RNA VL Anti-HMGB1 Abs r = −0.5 r = −0.49 P = 0.018 p < 0.0001n = 22 n = 61 r = coefficient of correlation; p < 0.05: >95% probabilitythat the two variables are correlated; n = number of patients in thestudy

As shown on FIG. 20A, there is an inverse correlation between HMGB1 andanti-HMGB1 antibody levels, indicating that HMGB1 production induces thesynthesis of anti-HMGB1 antibodies that neutralize HMGB1, as low levelsof HMGB1 are associated with high levels of antibodies.

FIG. 20B demonstrates that there is an inverse correlation betweenanti-HMGB1 antibodies and VL, suggesting that, due to the neutralizingactivity of anti-HMGB1 antibodies, the stimulating activity of HMGB1 onviral replication is suppressed by the antibodies.

These data argue for a beneficial effect of anti-HMGB1-based therapy inHIV+patients, which would lead to HMGB1 neutralization and therefore adecrease in the viral load.

MODIFICATIONS AND OTHER EMBODIMENTS

Various modifications and variations of the disclosed products,compositions, and methods as well as the concept of the invention willbe apparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed is not intended to be limitedto such specific embodiments. Various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the medical, immunological, biological, chemical or pharmacologicalarts or related fields are intended to be within the scope of thefollowing claims.

INCORPORATION BY REFERENCE

Each document, patent, patent application or patent publication cited byor referred to in this disclosure is incorporated by reference in itsentirety, especially with respect to the specific subject mattersurrounding the citation of the reference in the text. However, noadmission is made that any such reference constitutes background art andthe right to challenge the accuracy and pertinence of the citeddocuments is reserved.

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1. An in vitro method for quantitating the total antibodies specific forHigh mobility group box I (HMGB1) contained in a biological sampleobtained from a subject, comprising: a) treating the sample by an acidtreatment to dissociate the immune complexes found in the sample,preferably with Glycine 1.5M at a low pH; b) contacting said treatedbiological sample with native HMGB1 protein or derivatives thereof; andc) quantitating the total antibodies specific for High mobility groupbox I (HMGB1).
 2. An in vitro method for monitoring the humanimmunodeficiency virus (HIV) infection, in a biological sample obtainedfrom a subject who is known to be infected with HIV, comprisingquantitating antibodies specific for High mobility group box I (HMGB1),wherein said antibodies targeted for quantitation are either totalantibodies specific for HMGB1 or their circulating fraction (circulatingantibodies) or their immunologically complexed fraction.
 3. A methodaccording to claim 1 or 2, wherein the HMGB1 protein derivative isselected in the group consisting of a recombinant HMGB1, animmunologically reactive part of HMGB1, an immunologically reactive partof HMGB1 whose sequence is common to HMGB1 proteins of various originsand the recombinant BOXB from HMGB1 corresponding to the sequence commonto human and mouse of HMGB1.
 4. An in vitro method for monitoring theHIV viral load, in a biological sample obtained from a subject who isknown to be infected with HIV, comprising carrying out the method of anyone of claims 1 to 3, wherein the more the antibodies specific forHMGB1, the less the viral load.
 5. An in vitro method for monitoring theefficacy of a treatment directed against HIV infection, in a subjectinfected with HIV comprising carrying out the method of any one ofclaims 1 to 4 on samples obtained from said subject at different timesduring the treatment, and determining the efficacy of the treatmentgiven to the subject.
 6. An in vitro method according to claim 5,wherein the results obtained for samples obtained from the subject atdifferent times during the treatment are compared to the result obtainedwith a sample of the same subject prior to the initiation of thetreatment.
 7. An in vitro method according to any one of claims 1 to 6,wherein said biological sample obtained from a subject infected with HIVis blood, plasma, serum, saliva, peripheral blood mononuclear cells(PBMCs) or other body fluids or tissues.
 8. An in vitro prognosticmethod of either the state of progression of Acquired immune deficiencysyndrome (AIDS) or the state of progression toward AIDS, in a patientinfected with HIV, comprising carrying out the method of any one ofclaims 1 to 3 in a sample obtained from said patient after infection,and preferably during primary or acute infection or during chronicinfection, and wherein the more the level of antibodies specific forHMGB1, the less the risk to develop AIDS or an advanced state of AIDS.9. An in vitro method according to any one of claims 1 to 8, wherein thequantity of antibodies specific for HMGB1 is determined by ELISA usingthe High mobility group box I (HMGB1) protein or derivatives thereofcoated on a solid support.
 10. An in vitro method according to any ofclaims 2 to 9, wherein a step suitable for dissociation of immunologicalcomplexes formed with specific anti-HMGB1 antibodies is performed andthe quantitated antibodies specific for HMGB1 are total antibodiesspecific for HMGB1.
 11. An in vitro method according to any of claims 2to 9, wherein the quantitated antibodies specific for HMGB1 arecirculating antibodies.
 12. An in vitro method for monitoring HIVinfection in a subject infected with HIV comprising quantitating Highmobility group box I (HMGB1) protein contained in a biological sampleobtained from said subject, wherein said HMGB1 protein targeted forquantitation is either total HMGB1 or its circulating fraction(circulating HMGB1) or its immunologically complexed fraction.
 13. An invitro method according to claim 12 comprising contacting the biologicalsample from said subject infected with HIV, with antibodies thatimmunologically bind to High mobility group box I (HMGB1) protein. 14.An in vitro method according to any one of claims 11 to 13, wherein thequantitated HMGB1 protein is the circulating HMGB1 protein.
 15. An invitro method according to any one of claims 11 to 13, wherein thequantitated HMGB1 protein is the total HMGB1 protein, and the methodcomprises treating the sample by an acidic treatment in conditionsenabling immunological complexes formed with the HMGB1 protein todissociate.
 16. An in vitro method for monitoring the HIV viral load, ina biological sample obtained from a subject who is known to be infectedwith HIV, comprising carrying out the method of any one of claims 12 to15, wherein the more the HMGB1 protein, the more the viral load.
 17. Anin vitro method according to any one of claims 12 to 16, wherein saidquantitated HMGB1 is compared to the amount of HMGB1 from a biologicalsample obtained from a subject not infected with HIV, or to the amountof HMGB1 from a biological sample obtained from the same subject at adifferent time.
 18. An in vitro method according to any one of claims 12to 17, further comprising at least one diagnostic test to exclude orcontrol acute and/or chronic inflammation in said subject.
 19. An invitro method for monitoring the efficacy of a treatment directed againstHIV infection in a subject infected with HIV, comprising carrying outthe method of any one of claims 12 to 15, on samples obtained from saidsubject at different times during the treatment, and determining theefficacy of the treatment given to the subject, and optionally comparingthese results obtained with a sample of the same subject prior to theinitiation of the treatment.
 20. An in vitro prognostic method either ofthe state of progression of AIDS or of the state of progression towardsAIDS, in a patient infected with HIV, comprising carrying out the methodof any one of claims 12 to 15 in a sample obtained from said patientafter infection, and preferably during primary or acute infection orchronic infection, and wherein the more the level of HMGB1 protein, themore the risk to develop AIDS or an advanced state of AIDS.
 21. An invitro method according to any one of claims 12 to 20, wherein saidsample is blood, plasma, serum, saliva, PBMCs or other body fluids. 22.An in vitro method according to any one of claims 1 to 21, wherein saidHIV is HIV-1 or HIV-2.
 23. An in vitro method according to any one ofclaims 1 to 22, wherein said subject is a human being.
 24. An agent thatbinds to High mobility group box 1 protein (HMGB1) for use as a drug totreat HIV infection in human, optionally in combination with a furtheractive compound against HIV infection.
 25. An agent that binds to Highmobility group box 1 protein (HMGB1) for use as a drug to decrease theHIV reservoir cells, in a HIV-infected subject, optionally incombination with a further active compound against HIV infection.
 26. Anagent according to claim 25, wherein said targeted HIV reservoir cellsare cells originating from biological tissues, such as blood, solidtissues or mucosa, that are sensitive to and can be infected by HIV. 27.An agent according to claim 26, wherein said cells are cells from brain,liver, spleen, tonsils, nodes, gut-associated lymphoid tissue (GALT),are peripheral blood cells, lymphoid lineage cells such as T cellsespecially CD4 T cells, or are monocyte-derived cells such asmacrophages or dendritic cells.
 28. An agent according to claims 24 to27 which is chosen in the group consisting of: a) glycyrrhizin; b) anantibody specifically blocking HMGB1 or a fragment of such antibodywhich retains said ability to specifically block HMGB1, in particular amonoclonal antibody, a single-chain antibody, or a Fab, Fv, Fab₂fragment, wherein such antibody or fragment is preferably human orhumanized; and c) the isolated RAGE or fragment thereof able to bindHMGB1.
 29. A method for modulating human immunodeficiency virus (HIV)infection comprising contacting a subject infected by HIV with an agentthat binds to High mobility group box 1 protein (HMGB1), in particularan agent which inhibits natural killer (NK) cell dependent triggering ofHIV replication in a dendritic cell (DC).
 30. A method for decreasingHIV reservoir cells, comprising contacting a subject infected by HIVwith an agent that binds to High mobility group box 1 protein (HMGB1).31. A method according to claim 29 or 30, wherein said agent is chosenin the group consisting of: a) glycyrrhizin; b) an antibody specificallyblocking HMGB1 or a fragment of such antibody which retains said abilityto specifically block HMGB1, in particular a monoclonal antibody, asingle-chain antibody, or a Fab, Fv, Fab₂ fragment, wherein suchantibody or fragment is preferably human or humanized; and c) theisolated RAGE or fragment thereof able to bind HMGB1.
 32. A kit toquantitate the total antibodies specific for the High mobility group box1 protein (HMGB1) in a sample, comprising: a) native HMGB1 protein orderivatives thereof; b) an acidic dissociation solution suitable todissociate immunological HMGB1/anti-HMGB1 antibody complexes found inthe sample when taken from the patient; c) optionally, a neutralizationbuffer; and d) optionally, secondary antibodies binding to theHMGB1/specific antibodies complex.
 33. A kit to quantitate the totalHigh mobility group box 1 protein (HMGB1) in a sample, comprising: a) anantibody specific for the HMGB1 protein, or a fragment thereof able tobind the HMGB1 protein; b) an acidic dissociation solution suitable todissociate immunological HMGB1/anti-HMGB1 antibody complexes found inthe sample when taken from the patient; c) optionally, a neutralizationbuffer; and d) optionally, secondary antibodies binding to theHMGB1/specific antibodies complex.